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1.
Front Immunol ; 12: 686439, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34616392

RESUMO

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.


Assuntos
Anticorpos/metabolismo , Antígeno HLA-A2/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T Reguladores/transplante , Tolerância ao Transplante , Animais , Engenharia Celular , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Imunoterapia Adotiva , Masculino , Camundongos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 41(11): 2730-2739, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34587757

RESUMO

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Ácido N-Acetilneuramínico/administração & dosagem , Ácidos Neuramínicos/administração & dosagem , Placa Aterosclerótica , Ração Animal , Animais , Anticorpos/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Neuramínicos/imunologia , Ácidos Neuramínicos/metabolismo , Pan troglodytes , Receptores de LDL/genética , Receptores de LDL/metabolismo , Sialadenite/metabolismo , Sialadenite/patologia , Células THP-1
3.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576154

RESUMO

Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient's immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.


Assuntos
Linfócitos B/metabolismo , Edição de Genes/métodos , Animais , Anticorpos/metabolismo , Apresentação do Antígeno/genética , Apresentação do Antígeno/fisiologia , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos
4.
PLoS One ; 16(9): e0257147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34492074

RESUMO

Posttraumatic fibrotic scarring is a significant medical problem that alters the proper functioning of injured tissues. Current methods to reduce posttraumatic fibrosis rely on anti-inflammatory and anti-proliferative agents with broad intracellular targets. As a result, their use is not fully effective and may cause unwanted side effects. Our group previously demonstrated that extracellular collagen fibrillogenesis is a valid and specific target to reduce collagen-rich scar buildup. Our previous studies showed that a rationally designed antibody that binds the C-terminal telopeptide of the α2(I) chain involved in the aggregation of collagen molecules limits fibril assembly in vitro and reduces scar formation in vivo. Here, we have utilized a clinically relevant arthrofibrosis model to study the broad mechanisms of the anti-scarring activity of this antibody. Moreover, we analyzed the effects of targeting collagen fibril formation on the quality of healed joint tissues, including the posterior capsule, patellar tendon, and subchondral bone. Our results show that blocking collagen fibrillogenesis not only reduces collagen content in the scar, but also accelerates the remodeling of healing tissues and changes the collagen fibrils' cross-linking. In total, this study demonstrated that targeting collagen fibrillogenesis to limit arthrofibrosis affects neither the quality of healing of the joint tissues nor disturbs vital tissues and organs.


Assuntos
Colágenos Fibrilares/metabolismo , Artropatias/patologia , Artropatias/fisiopatologia , Articulações/fisiopatologia , Animais , Anticorpos/metabolismo , Biomarcadores/sangue , Células CHO , Calcificação Fisiológica , Cricetulus , Modelos Animais de Doenças , Feminino , Fibrose , Cápsula Articular/metabolismo , Cápsula Articular/patologia , Cápsula Articular/fisiopatologia , Masculino , Coelhos , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo
5.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443500

RESUMO

Prostaglandins are a group of important cell-signaling molecules involved in the regulation of ovarian maturation, oocyte development, egg laying and associated behaviors in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for PGE2 biosynthesis and its interference by drugs were not investigated previously in the ovary of ticks. The present study was undertaken to assess the modulation of the PGE2-mediated pathway in the eclosion blocking effect of flumethrin and terpenoid subfraction isolated from Artemisia nilagirica in Rhipicephalus annulatus ticks. The acaricidal activities and chemical profiling of the terpenoid subfraction were performed. The localization of the cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) enzymes and the quantification of PGE2 in the ovaries of the ticks treated with methanol (control), flumethrin and terpenoid subfraction were also undertaken. In addition, the vitellogenin concentration in hemolymph was also assayed. Both flumethrin and the terpenoid subfraction of A. nilagirica elicited a concentration-dependent inhibition of fecundity and blocking of hatching of the eggs. The COX1 could not be detected in the ovaries of treated and control ticks, while there was no significant difference observed in the concentration of vitellogenin (Vg) in them. The presence of PGES in the oocytes of control ticks was confirmed while the immunoreactivities against PGES were absent in the vitellogenic oocytes of ticks treated with flumethrin and terpenoid subfraction. The levels of PGE2 were below the detection limit in the ovaries of the flumethrin-treated ticks, while it was significantly lower in the ovaries of the terpenoid subfraction-treated ticks. Hence, the prostaglandin E synthase and PGE2 were identified as very important mediators for the signaling pathway for ovarian maturation and oviposition in ticks. In addition, the key enzyme for prostaglandin biosynthesis, PGES and the receptors for PGE2 can be exploited as potential drug targets for tick control. The detection of PGES by immunohistochemistry and quantification of PGE2 by LC-MSMS can be employed as valuable tools for screening newer compounds for their eclosion blocking acaricidal effects.


Assuntos
Artemisia/química , Dinoprostona/metabolismo , Piretrinas/farmacologia , Rhipicephalus/efeitos dos fármacos , Terpenos/isolamento & purificação , Terpenos/farmacologia , Animais , Anticorpos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hemolinfa/metabolismo , Imersão , Ovário/efeitos dos fármacos , Ovário/enzimologia , Peroxidase/metabolismo , Prostaglandina-E Sintases/metabolismo , Vitelogeninas/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445351

RESUMO

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas/metabolismo , Análise de Célula Única/métodos , Anticorpos/metabolismo , Comunicação Celular , Imunofluorescência/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Formaldeído/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Técnicas Imunoenzimáticas/métodos , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Inclusão em Parafina , Proteínas/análise , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
7.
Biomed Res Int ; 2021: 6676107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368354

RESUMO

The present study is aimed at profiling circulating exosome-derived microRNAs (miRNAs/miRs) from patients with dermatomyositis (DM), in particular those complicated with interstitial lung disease (ILD) with anti-melanoma differentiation-associated protein 5 (MDA5) antibody-positive. Fifteen participants were enrolled, including five patients with DM complicated with ILDs prior to treatment with circulating anti-MDA5 antibody-positive status [DM-ILD-MDA5 Ab(+)], five DM patients without ILDs who were negative for 16 detectable myositis-specific antibodies [DM-nonILD-MSA16(-)], and five age- and gender-matched healthy donor controls (HCs). The characteristics of the exosomes extracted by Ribo™ Exosome Isolation Reagent were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Differentially expressed miRNAs, determined by next-generation deep sequencing, were identified through the criteria of ∣log2 fold change | ≥1 and P < 0.01. A total of 38 miRNAs were significantly upregulated in exosomes from patients with DM-ILD-MDA5 Ab(+) compared to those from HC, while 21 miRNAs were significantly downregulated. Compared to exosomes derived from patients with DM-nonILD-MSA16(-), 51 miRNAs were significantly upregulated and 33 miRNAs were significantly downregulated from patients with DM-ILD-MDA5 Ab(+). A total of 73 exosomal miRNAs were significantly differentially expressed between DM-nonILD-MSA16(-) and HC. In particular, two miRNAs, Homo sapiens- (hsa-) miR-4488 and hsa-miR-1228-5p, were common differentially expressed miRNAs among three comparisons. GO and KEGG analyses suggested that several pathways may contribute the pathogenesis of DM-ILD-MDA5 Ab(+) and DM-nonILD-MSA16(-), while PPI network analysis of hsa-miR-4488 and hsa-miR-1228-5p indicated that their predicted target genes, DExD-box helicase 39B and MDM2, may be involved in the mechanisms of DM-ILD-MDA5 Ab(+).


Assuntos
Anticorpos/metabolismo , Dermatomiosite/sangue , Exossomos/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Doenças Pulmonares Intersticiais/sangue , MicroRNAs/sangue , Adulto , Biomarcadores/sangue , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Dermatomiosite/complicações , Dermatomiosite/genética , Exossomos/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/genética , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Sci Rep ; 11(1): 16951, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417497

RESUMO

T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry-leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Fosfosserina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Anticorpos/metabolismo , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinases TOR/metabolismo
9.
Biochem Biophys Res Commun ; 573: 35-41, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34388452

RESUMO

Fusion of a target-specific peptide to a full-length antibody (Ab) can result in a peptide-Ab fusion protein with additional specificity and enhanced activity. We recently developed an intracellular pan-RAS-targeting cytosol-penetrating antibody, RT22-ep59, in which a tumor-specific targeting ability was achieved via the fusion of an epithelial cell adhesion molecule (EpCAM) targeting cyclic peptide (ep133). Here, the aim was to enhance EpCAM-mediated endocytosis and tumor accumulation of the peptide-fused RAS-targeting Ab. Accordingly, we engineered a cyclic peptide (from ep133) that has stronger affinity for EpCAM by using yeast surface display technology and then rationally designed cyclic peptides in the Ab-fused form to enhance colloidal stability. The finally engineered EpCAM-targeting cyclic peptide (ep6)-fused Ab, ep6Ras37, has ∼10-fold stronger affinity (KD ≈ 1.9 nM) for EpCAM than that of RT22-ep59, without deterioration of biophysical properties. Compared with the parental antibody (RT22-ep59), ep6Ras37 more efficiently reached the cytosol of EpCAM-expressing cells and showed greater preferential tumor homing and accumulation in mice bearing EpCAM-expressing LoVo xenograft tumors. Thus, the high-affinity EpCAM-targeting peptide ensures efficient cellular internalization and better tumor accumulation of the peptide-fused Ab.


Assuntos
Anticorpos/metabolismo , Neoplasias do Colo/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Peptídeos Cíclicos/metabolismo , Engenharia de Proteínas , Animais , Anticorpos/química , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos Cíclicos/química , Distribuição Tecidual
10.
Acc Chem Res ; 54(18): 3576-3592, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34406761

RESUMO

Most therapeutic peptides available on the market today are naturally occurring hormones or protein fragments that were serendipitously discovered to possess therapeutic effects. However, the limited repertoire of available natural resources presents difficulties for the development of new peptide drug candidates. Traditional peptides possess several shortcomings that must be addressed for biomedical applications, including relatively low affinity or specificity toward biological targets compared to antibody- and protein scaffold-based affinity molecules, poor in vivo stability owing to rapid enzymatic degradation, and rapid clearance from circulation owing to their small size. Going forward, it will be increasingly important for scientists to develop novel classes of high-affinity and -specificity peptides against desired targets that mitigate these limitations while remaining compatible with pharmaceutical manufacturing processes. Recently, several highly constrained, artificial cyclic peptides have emerged as platforms capable of generating high-affinity peptide binders against various disease-associated protein targets by combining with phage or mRNA display method, some of which have entered clinical trials. In contrast, although linear peptides are relatively easy to synthesize cost-effectively and modify site-specifically at either N- or C-termini compared to cyclic peptides, there have been few linear peptide-based platforms that can provide high-affinity and -specificity peptide binders.In this Account, we describe the creation and development of a novel class of high-affinity peptides, termed "aptide"-from the Latin word "aptus" meaning "to fit" and "peptide"-and summarize their biomedical applications. In the first part, we consider the design and creation of aptides, with a focus on their unique structural features and binding mode, and address screening and identification of target protein-specific aptides. We also discuss advantages of the aptide platform over ordinary linear peptides lacking preorganized structures in terms of the affinity and specificity of identified peptide binders against target molecules. In the second part, we describe the potential biomedical applications of various target-specific aptides, ranging from imaging and therapy to theranostics, according to the types of aptides and diseases. We show that certain aptides can not only bind to a target protein but also inhibit its biological function, thereby showing potential as therapeutics per se. Further, aptides specific for cancer-associated protein antigens can be used as escort molecules or targeting ligands for delivery of chemotherapeutics, cytokine proteins, and nanomedicines, such as liposomes and magnetic particles, to tumors, thereby substantially improving therapeutic effects. Finally, we present a strategy capable of overcoming the critical issue of short blood circulation time associated with most peptides by constructing a hybrid system between an aptide and a hapten cotinine-specific antibody.


Assuntos
Nanomedicina , Peptídeos/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Retinopatia Diabética/tratamento farmacológico , Humanos , Cinética , Magnetismo , Camundongos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Peptídeos/química , Peptídeos/uso terapêutico , Estrutura Terciária de Proteína , Fator de Transcrição STAT3/química , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/química
11.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208575

RESUMO

Due to their high specificity, monoclonal antibodies have been widely investigated for their application in drug delivery to the central nervous system (CNS) for the treatment of neurological diseases such as stroke, Alzheimer's, and Parkinson's disease. Research in the past few decades has revealed that one of the biggest challenges in the development of antibodies for drug delivery to the CNS is the presence of blood-brain barrier (BBB), which acts to restrict drug delivery and contributes to the limited uptake (0.1-0.2% of injected dose) of circulating antibodies into the brain. This article reviews the various methods currently used for antibody delivery to the CNS at the preclinical stage of development and the underlying mechanisms of BBB penetration. It also describes efforts to improve or modulate the physicochemical and biochemical properties of antibodies (e.g., charge, Fc receptor binding affinity, and target affinity), to adapt their pharmacokinetics (PK), and to influence their distribution and disposition into the brain. Finally, a distinction is made between approaches that seek to modify BBB permeability and those that use a physiological approach or antibody engineering to increase uptake in the CNS. Although there are currently inherent difficulties in developing safe and efficacious antibodies that will cross the BBB, the future prospects of brain-targeted delivery of antibody-based agents are believed to be excellent.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Animais , Anticorpos/administração & dosagem , Anticorpos/efeitos adversos , Anticorpos/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Transporte Biológico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Vias de Administração de Medicamentos , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Permeabilidade , Agregados Proteicos , Agregação Patológica de Proteínas , Engenharia de Proteínas , Distribuição Tecidual
12.
J Immunol ; 207(3): 755-764, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34321286

RESUMO

Recent developments in genome editing and delivery systems have opened new possibilities for B cell gene therapy. CRISPR-Cas9 nucleases have been used to introduce transgenes into B cell genomes for subsequent secretion of exogenous therapeutic proteins from plasma cells and to program novel B cell Ag receptor specificities, allowing for the generation of desirable Ab responses that cannot normally be elicited in animal models. Genome modification of B cells or their progenitor, hematopoietic stem cells, could potentially substitute Ab or protein replacement therapies that require multiple injections over the long term. To date, B cell editing using CRISPR-Cas9 has been solely employed in preclinical studies, in which cells are edited ex vivo. In this review, we discuss current B cell engineering efforts and strategies for the eventual safe and economical adoption of modified B cells into the clinic, including in vivo viral delivery of editing reagents to B cells.


Assuntos
Linfócitos B/fisiologia , Terapia Genética/tendências , Receptores de Antígenos de Linfócitos B/genética , Animais , Anticorpos/genética , Anticorpos/metabolismo , Sistemas CRISPR-Cas , Epitopos , Engenharia Genética , Humanos , Imunoterapia
13.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34232287

RESUMO

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.


Assuntos
DNA/metabolismo , Sequências Repetidas Invertidas , RNA de Cadeia Dupla/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/metabolismo , Coloração e Rotulagem/métodos , Anticorpos/química , Anticorpos/metabolismo , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Clonagem Molecular , DNA/química , DNA/ultraestrutura , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Hibridização de Ácido Nucleico , Imagem Óptica/métodos , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Ribonuclease H/genética
14.
Cells ; 10(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065953

RESUMO

Macrophages play a key role in induction of inflammatory responses. These inflammatory responses are mostly considered to be instigated by activation of pattern recognition receptors (PRRs) or cytokine receptors. However, recently it has become clear that also antibodies and pentraxins, which can both activate Fc receptors (FcRs), induce very powerful inflammatory responses by macrophages that can even be an order of magnitude greater than PRRs. While the physiological function of this antibody-dependent inflammation (ADI) is to counteract infections, undesired activation or over-activation of this mechanism will lead to pathology, as observed in a variety of disorders, including viral infections such as COVID-19, chronic inflammatory disorders such as Crohn's disease, and autoimmune diseases such as rheumatoid arthritis. In this review we discuss how physiological ADI provides host defense by inducing pathogen-specific immunity, and how erroneous activation of this mechanism leads to pathology. Moreover, we will provide an overview of the currently known signaling and metabolic pathways that underlie ADI, and how these can be targeted to counteract pathological inflammation.


Assuntos
Anticorpos/metabolismo , Proteína C-Reativa/metabolismo , Inflamação/imunologia , Componente Amiloide P Sérico/metabolismo , Anticorpos/imunologia , Proteína C-Reativa/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Inflamação/metabolismo , Inflamação/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Redes e Vias Metabólicas/imunologia , Receptores Fc/metabolismo , Componente Amiloide P Sérico/imunologia , Transdução de Sinais/imunologia
15.
Nat Protoc ; 16(7): 3522-3546, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34089021

RESUMO

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.


Assuntos
Dosagem de Genes , Imunoensaio/métodos , Proteínas/metabolismo , Análise de Célula Única , Anticorpos/metabolismo , Calibragem , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Dados , Ouro/química , Humanos , Proteínas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Coloração e Rotulagem
16.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066527

RESUMO

Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vß8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.


Assuntos
Optogenética , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Anticorpos/metabolismo , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Meia-Vida , Humanos , Células Jurkat , Ligantes , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/metabolismo
18.
PLoS One ; 16(6): e0252577, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34133431

RESUMO

Exosomes are a class of extracellular vesicles (EVs) that are mediators of normal intercellular communication, but exosomes are also used by tumor cells to promote oncogenesis and metastasis. Complement factor H (CFH) protects host cells from attack and destruction by the alternative pathway of complement-dependent cytotoxicity (CDC). Here we show that CFH can protect exosomes from complement-mediated lysis and phagocytosis. CFH was found to be associated with EVs from a variety of tumor cell lines as well as EVs isolated from the plasma of patients with metastatic non-small cell lung cancer. Higher levels of CFH-containing EVs correlated with higher metastatic potential of cell lines. GT103, a previously described antibody to CFH that preferentially causes CDC of tumor cells, was used to probe the susceptibility of tumor cell-derived exosomes to destruction. Exosomes were purified from EVs using CD63 beads. Incubation of GT103 with tumor cell-derived exosomes triggered exosome lysis primarily by the classical complement pathway as well as antibody-dependent exosome phagocytosis by macrophages. These results imply that GT103-mediated exosome destruction can be triggered by antibody Fc-C1q interaction (in the case of lysis), and antibody-Fc receptor interactions (in the case of phagocytosis). Thus, this work demonstrates CFH is expressed on tumor cell derived exosomes, can protect them from complement lysis and phagocytosis, and that an anti-CFH antibody can be used to target tumor-derived exosomes for exosome destruction via innate immune mechanisms. These findings suggest that a therapeutic CFH antibody has the potential to inhibit tumor progression and reduce metastasis promoted by exosomes.


Assuntos
Fator H do Complemento/metabolismo , Exossomos/metabolismo , Macrófagos/imunologia , Fagocitose/fisiologia , Anticorpos/imunologia , Anticorpos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Fator H do Complemento/imunologia , Humanos , Imunidade Inata , Leucócitos Mononucleares/citologia , Neoplasias Pulmonares/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Estadiamento de Neoplasias , Receptores de Complemento/metabolismo , Células Tumorais Cultivadas
19.
Sci Rep ; 11(1): 12795, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140538

RESUMO

The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex.


Assuntos
Anticorpos/metabolismo , Cromatina/metabolismo , Epigênese Genética , Acetilação , Acilação , Sequência de Aminoácidos , Animais , Ácido Butírico/metabolismo , Bovinos , Células HeLa , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Peptídeos/química , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Soroalbumina Bovina/química
20.
Front Immunol ; 12: 651656, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936072

RESUMO

Although immune dysfunction is a key feature of coronavirus disease 2019 (COVID-19), the metabolism-related mechanisms remain elusive. Here, by reanalyzing single-cell RNA sequencing data, we delineated metabolic remodeling in peripheral blood mononuclear cells (PBMCs) to elucidate the metabolic mechanisms that may lead to the progression of severe COVID-19. After scoring the metabolism-related biological processes and signaling pathways, we found that mono-CD14+ cells expressed higher levels of glycolysis-related genes (PKM, LDHA and PKM) and PPP-related genes (PGD and TKT) in severe patients than in mild patients. These genes may contribute to the hyperinflammation in mono-CD14+ cells of patients with severe COVID-19. The mono-CD16+ cell population in COVID-19 patients showed reduced transcription levels of genes related to lysine degradation (NSD1, KMT2E, and SETD2) and elevated transcription levels of genes involved in OXPHOS (ATP6V1B2, ATP5A1, ATP5E, and ATP5B), which may inhibit M2-like polarization. Plasma cells also expressed higher levels of the OXPHOS gene ATP13A3 in COVID-19 patients, which was positively associated with antibody secretion and survival of PCs. Moreover, enhanced glycolysis or OXPHOS was positively associated with the differentiation of memory B cells into plasmablasts or plasma cells. This study comprehensively investigated the metabolic features of peripheral immune cells and revealed that metabolic changes exacerbated inflammation in monocytes and promoted antibody secretion and cell survival in PCs in COVID-19 patients, especially those with severe disease.


Assuntos
COVID-19/imunologia , Glicólise/genética , Lisina/metabolismo , Monócitos/metabolismo , Análise de Célula Única/métodos , Adenosina Trifosfatases/sangue , Adenosina Trifosfatases/genética , Anticorpos/metabolismo , COVID-19/metabolismo , COVID-19/fisiopatologia , Bases de Dados Genéticas , Proteínas Ligadas por GPI/metabolismo , Ontologia Genética , Hematopoese/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Receptores de Lipopolissacarídeos/metabolismo , Lisina/genética , Proteínas de Membrana Transportadoras/sangue , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Monócitos/imunologia , Monócitos/patologia , Fosforilação Oxidativa , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transcriptoma/genética
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