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1.
J Biomed Sci ; 31(1): 29, 2024 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-38491519

RESUMO

Synthetic antibodies (Abs) represent a category of artificial proteins capable of closely emulating the functions of natural Abs. Their in vitro production eliminates the need for an immunological response, streamlining the process of Ab discovery, engineering, and development. These artificially engineered Abs offer novel approaches to antigen recognition, paratope site manipulation, and biochemical/biophysical enhancements. As a result, synthetic Abs are fundamentally reshaping conventional methods of Ab production. This mirrors the revolution observed in molecular biology and genomics as a result of deep sequencing, which allows for the swift and cost-effective sequencing of DNA and RNA molecules at scale. Within this framework, deep sequencing has enabled the exploration of whole genomes and transcriptomes, including particular gene segments of interest. Notably, the fusion of synthetic Ab discovery with advanced deep sequencing technologies is redefining the current approaches to Ab design and development. Such combination offers opportunity to exhaustively explore Ab repertoires, fast-tracking the Ab discovery process, and enhancing synthetic Ab engineering. Moreover, advanced computational algorithms have the capacity to effectively mine big data, helping to identify Ab sequence patterns/features hidden within deep sequencing Ab datasets. In this context, these methods can be utilized to predict novel sequence features thereby enabling the successful generation of de novo Ab molecules. Hence, the merging of synthetic Ab design, deep sequencing technologies, and advanced computational models heralds a new chapter in Ab discovery, broadening our comprehension of immunology and streamlining the advancement of biological therapeutics.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Sítios de Ligação de Anticorpos
2.
Mol Biol Rep ; 51(1): 134, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38236361

RESUMO

Synthetic antibodies (Abs) are a class of engineered proteins designed to mimic the functions of natural Abs. These are produced entirely in vitro, eliminating the need for an immune response. As such, synthetic Abs have transformed the traditional methods of raising Abs. Likewise, deep sequencing technologies have revolutionized genomics and molecular biology. These enable the rapid and cost-effective sequencing of DNA and RNA molecules. They have allowed for accurate and inexpensive analysis of entire genomes and transcriptomes. Notably, via deep sequencing it is now possible to sequence a person's entire B-cell receptor immune repertoire, termed BCR sequencing. This procedure allows for big data explorations of natural Abs associated with an immune response. Importantly, the identified sequences have the ability to improve the design and engineering of synthetic Abs by offering an initial sequence framework for downstream optimizations. Additionally, machine learning algorithms can be introduced to leverage the vast amount of BCR sequencing datasets to rapidly identify patterns hidden in big data to effectively make in silico predictions of antigen selective synthetic Abs. Thus, the convergence of BCR sequencing, machine learning, and synthetic Ab development has effectively promoted a new era in Ab therapeutics. The combination of these technologies is driving rapid advances in precision medicine, diagnostics, and personalized treatments.


Assuntos
Anticorpos , Receptores de Antígenos de Linfócitos B , Humanos , Receptores de Antígenos de Linfócitos B/genética , Anticorpos/genética , Algoritmos , Big Data , Genômica
3.
Bioconjug Chem ; 31(1): 16-27, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31789501

RESUMO

Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Animais , Materiais Biocompatíveis/química , Fluorescência , Humanos , Modelos Moleculares , Anticorpos de Cadeia Única/química
4.
Immunity ; 34(1): 50-60, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21236706

RESUMO

Self-reactive T cell clones that escape negative selection are either deleted or rendered functionally unresponsive (anergic), thus preventing them from propagating host tissue damage. By using an in vivo model, we investigated molecular mechanisms for T cell tolerance, finding that despite a characteristic inability to generate effector cytokine proteins, self-reactive T cells express large amounts of cytokine mRNAs. This disconnect between cytokine message and protein was not observed in T cells mounting productive responses to foreign antigens but, instead, was seen only in those responding to self, where the block in protein translation was shown to involve conserved AU-rich elements within cytokine 3'UTRs. These studies reveal that translation of abundant cytokine mRNAs is limited in self-reactive T cells and, thus, identify posttranscriptional silencing of antigen-driven gene expression as a key mechanism underlying the anergic phenotype of self-reactive T cells.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Regiões 3' não Traduzidas/genética , Transferência Adotiva , Animais , Autoimunidade , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal , Citocinas/genética , Citocinas/imunologia , Tolerância Imunológica , Camundongos , Camundongos Transgênicos , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Complexo de Inativação Induzido por RNA/imunologia , Elementos de Resposta/genética
5.
Cell Biol Int ; 44(6): 1267-1282, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32162758

RESUMO

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that is part of the family of tyrosine kinase receptors. The binding of EGFR to its cognate ligands leads to its autophosphorylation and subsequent activation of the signal transduction pathways involved in regulating cellular proliferation, differentiation, and survival. Accordingly, this receptor carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. Correspondingly, the loss of EGFR regulation results in many human diseases, with the most notable cancer. This receptor is overexpressed and/or mutated in multiple epithelial-derived tumors, and associated with poor prognosis and survival in cancer patients. Here, we discuss in detail the role of EGFR in specific epithelial-derived cancer pathologies; these include lung cancer, colorectal cancer, and squamous cell carcinomas. The development of multiple anticancer agents against EGFR diminished the progression and metastasis of tumors. Some of the most versatile therapeutic anti-EGFR agents include the monoclonal antibodies (mAbs), demonstrating success in clinical settings when used in combination with cytotoxic treatments, such as chemotherapy and/or radiation. We thus discuss the development and application of two of the most notable therapeutic mAbs, cetuximab, and panitumumab, currently utilized in various EGFR-related epithelial cancers.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas , Neoplasias Colorretais , Neoplasias Pulmonares , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Panitumumabe/uso terapêutico
6.
Mol Biol Rep ; 47(10): 8037-8048, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32990903

RESUMO

The Eph (erythropoietin-producing human hepatocellular) receptors form the largest known subfamily of receptor tyrosine kinases. These receptors interact with membrane-bound ephrin ligands via direct cell-cell interactions resulting in bi-directional activation of signal pathways. Importantly, the Eph receptors play critical roles in embryonic tissue organization and homeostasis, and in the maintenance of adult processes such as long-term potentiation, angiogenesis, and stem cell differentiation. The Eph receptors also display properties of both tumor promoters and suppressors depending on the cellular context. Characterization of EphA2 receptor in regard to EphA2 dysregulation has revealed associations with various pathological processes, especially cancer. The analysis of various tumor types generally identify EphA2 receptor as overexpressed and/or mutated, and for certain types of cancers EphA2 is linked with poor prognosis and decreased patient survival. Thus, here we highlight the role of EphA2 in malignant tissues that are specific to cancer; these include glioblastoma multiforme, prostate cancer, ovarian and uterine cancers, gastric carcinoma, melanoma, and breast cancer. Due to its large extracellular domain, therapeutic targeting of EphA2 with monoclonal antibodies (mAbs), which may function as inhibitors of ligand activation or as molecular agonists, has been an oft-attempted strategy. Therefore, we review the most current mAb-based therapies against EphA2 expressing cancers currently in pre-clinical and/or clinical stages. Finally, we discuss the latest peptides and cyclical-peptides that function as selective agonists for EphA2 receptor.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Efrina-A2/imunologia , Imunoterapia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Antineoplásicos Imunológicos/imunologia , Efrina-A2/antagonistas & inibidores , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/patologia , Receptor EphA2
7.
Mol Biol Rep ; 47(7): 5523-5533, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32621117

RESUMO

The erythropoietin-producing human hepatocellular (Eph) receptors are transmembrane glycoprotein members of the tyrosine kinase receptors family. The Ephs may bind to various ephrin ligands resulting in the phosphorylation of their tyrosine kinase domain and the activation of the Eph receptor. In this review we focus on EphA3, one receptor of the 14 different Ephs, as it carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. The loss of EphA3 regulation is correlated with various human malignancies, the most notable being cancer. This receptor is overexpressed and/or mutated in multiple tumors, and is also associated with poor prognosis and decreased survival in patients. Here we highlight the role of EphA3 in normal and malignant tissues that are specific to cancer; these include hematologic disorders, gastric cancer, glioblastoma multiforme, colorectal cancer, lung cancer, renal cell carcinoma, and prostate cancer. Moreover, various anticancer agents against EphA3 have been developed to either inhibit its kinase domain activity or to function as agonists. Thus, we examine the most potent small molecule drugs and mAb-based therapeutics against EphA3 that are currently in pre-clinical or clinical stages.


Assuntos
Neoplasias/genética , Receptor EphA3/genética , Receptor EphA3/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Renais , Neoplasias Colorretais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma , Humanos , Neoplasias Renais , Neoplasias Pulmonares , Masculino , Neoplasias/tratamento farmacológico , Fosforilação , Neoplasias da Próstata , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo
8.
J Cell Sci ; 130(15): 2644-2653, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28615413

RESUMO

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.


Assuntos
Técnicas Biossensoriais/métodos , Imunofluorescência/métodos , Anticorpos de Cadeia Única/química , Linhagem Celular , Humanos
9.
Biotechnol Bioeng ; 111(3): 475-84, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24122476

RESUMO

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins-Protein A or Protein G-and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/metabolismo , Imunoensaio/métodos , Proteínas Recombinantes de Fusão/análise , Coloração e Rotulagem/métodos , Fluorescência , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética
10.
Mol Biotechnol ; 2024 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-38308755

RESUMO

Synthetic antibodies (Abs) represent a category of engineered proteins meticulously crafted to replicate the functions of their natural counterparts. Such Abs are generated in vitro, enabling advanced molecular alterations associated with antigen recognition, paratope site engineering, and biochemical refinements. In a parallel realm, deep sequencing has brought about a paradigm shift in molecular biology. It facilitates the prompt and cost-effective high-throughput sequencing of DNA and RNA molecules, enabling the comprehensive big data analysis of Ab transcriptomes, including specific regions of interest. Significantly, the integration of artificial intelligence (AI), based on machine- and deep- learning approaches, has fundamentally transformed our capacity to discern patterns hidden within deep sequencing big data, including distinctive Ab features and protein folding free energy landscapes. Ultimately, current AI advances can generate approximations of the most stable Ab structural configurations, enabling the prediction of de novo synthetic Abs. As a result, this manuscript comprehensively examines the latest and relevant literature concerning the intersection of deep sequencing big data and AI methodologies for the design and development of synthetic Abs. Together, these advancements have accelerated the exploration of antibody repertoires, contributing to the refinement of synthetic Ab engineering and optimizations, and facilitating advancements in the lead identification process.

11.
Eur J Immunol ; 42(9): 2322-8, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22684943

RESUMO

In helper T cells, IL-13 is traditionally considered a Th2-type cytokine that is coexpressed with IL-4. Using mouse models of immunization and autoimmunity, we demonstrate that IL-13 is frequently uncoupled from IL-4, and that it can be produced by both IFN-γ(+) Th1 cells and IL-17(+) Th17 cells. We report that these IL-13-producing Th1 and Th17 cells are distinct from classical IL-4(+) Th2 cells and that they are relatively common, appearing in the context of both protective and pathogenic T-cell responses. We also demonstrate that IL-13 and Th2-type cytokines can have important consequences in Th1- and Th17-dominated settings, such as lymphopenia-induced autoimmune disease, where they can be either pro- or anti-inflammatory, depending on whether they act on innate or adaptive immune cells. Taken together, our studies indicate that IL-13 production is more widespread than previously appreciated and that blocking this cytokine may have therapeutic benefits even in settings where traditional IL-4-driven Th2-type responses are not evident.


Assuntos
Antígenos/imunologia , Autoimunidade/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Doenças Autoimunes/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Th2/imunologia
12.
J Immunol ; 186(8): 4668-73, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21402892

RESUMO

T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4(+) T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4(+) T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4(+) T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Células Th1/metabolismo , Células Th17/metabolismo
13.
Proc Natl Acad Sci U S A ; 107(42): 18085-90, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20921406

RESUMO

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.


Assuntos
Tolerância Imunológica , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Animais , Células Apresentadoras de Antígenos/imunologia , Citometria de Fluxo , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos BALB C
14.
Blood ; 116(17): 3171-9, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20644121

RESUMO

After the identification of discrete relapse-risk categories in patients with acute promyelocytic leukemia (APL) receiving all-trans retinoic and idarubicin (AIDA)-like therapies, the Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) designed a protocol for newly diagnosed APL (AIDA-2000) in which postremission treatment was risk-adapted. Patients with low/intermediate risk received remission at 3 anthracycline-based consolidation courses, whereas high-risk patients received the same schedule as in the previous, non-risk-adapted AIDA-0493 trial including cytarabine. In addition, all patients in the AIDA-2000 received all-trans retinoic acid (ATRA) for 15 days during each consolidation. After induction, 600 of 636 (94.3%) and 420 of 445 (94.4%) patients achieved complete remission in the AIDA-0493 and AIDA-2000, respectively. The 6-year overall survival and cumulative incidence of relapse (CIR) rates were 78.1% versus 87.4% (P = .001) and 27.7% versus 10.7% (P < .0001). Significantly lower CIR rates for patients in the AIDA-2000 were most evident in the high-risk group (49.7% vs 9.3%, respectively, P < .0001). Our data confirm that anthracycline-based consolidation is at least equally effective as cytarabine-containing regimens for low-/intermediate-risk patients and suggest that a risk-adapted strategy including ATRA for consolidation improves outcome in newly diagnosed APL. Furthermore, our results highlight the role of cytarabine coupled to anthracyclines and ATRA during consolidation in the high-risk group.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Idarubicina/uso terapêutico , Leucemia Promielocítica Aguda/tratamento farmacológico , Tretinoína/uso terapêutico , Adolescente , Adulto , Antraciclinas/administração & dosagem , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Citarabina/administração & dosagem , Citarabina/uso terapêutico , Humanos , Idarubicina/administração & dosagem , Leucemia Promielocítica Aguda/diagnóstico , Pessoa de Meia-Idade , Indução de Remissão , Tretinoína/administração & dosagem , Adulto Jovem
15.
J Immunol ; 185(11): 6461-71, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20974984

RESUMO

Given the association with autoimmune disease, there is great interest in defining cellular factors that limit overactive or misdirected Th17-type inflammation. Using in vivo and in vitro models, we investigated the molecular mechanisms for cytokine-mediated inhibition of Th17 responses, focusing on the role of STAT1 and T-bet in this process. These studies demonstrate that, during systemic inflammation, STAT1- and T-bet-deficient T cells each exhibit a hyper-Th17 phenotype relative to wild-type controls. However, IL-17 production was greater in the absence of T-bet, and when both STAT1 and T-bet were deleted, there was no further increase, with the double-deficient cells instead behaving more like STAT1-deficient counterparts. Similar trends were observed during in vitro priming, with production of Th17-type cytokines greater in T-bet(-/-) T cells than in either STAT1(-/-) or STAT1(-/-) T-bet(-/-) counterparts. The ability of IFN-γ and IL-27 to suppress Th17 responses was reduced in T-bet-deficient cells, and most importantly, ectopic T-bet could suppress signature Th17 gene products, including IL-17A, IL-17F, IL-22, and retinoic acid-related orphan receptor γT, even in STAT1-deficient T cells. Taken together, these studies formally establish that, downstream of IFN-γ, IL-27, and likely all STAT1-activating cytokines, there are both STAT1 and T-bet-dependent pathways capable of suppressing Th17 responses.


Assuntos
Citocinas/fisiologia , Interleucina-17/antagonistas & inibidores , Fator de Transcrição STAT1/fisiologia , Proteínas com Domínio T/fisiologia , Células Th1/imunologia , Células Th1/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Células Cultivadas , Citocinas/metabolismo , Imunofenotipagem , Mediadores da Inflamação/fisiologia , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/fisiologia , Interleucina-17/biossíntese , Interleucinas/fisiologia , Linfopenia/imunologia , Linfopenia/metabolismo , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/transplante
16.
J Immunol ; 184(1): 30-4, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19949064

RESUMO

The possibility that effector T cells can be converted into forkhead box P3(+) regulatory T cells (Tregs) has potential therapeutic implications. To analyze the relationship between Th1 effectors and Tregs, we have used a model of systemic autoimmunity in which both effector and Tregs arise from a single population specific for a transgene-encoded systemic protein. In vitro, the presence of IFN-gamma inhibits Treg generation during activation. Using IFN-gamma reporter mice, we demonstrate that IFN-gamma-producing cells tend not to develop into Tregs, and Th1 priming of T cells prior to cell transfer limits the number of forkhead box P3(+) T cells generated in vivo. Moreover, transfer of IFN-gamma(-/-) or STAT1(-/-) T cells resulted in an increase in the number of Tregs. These data support a role for Th1 effector molecules and transcription factors in the control of peripheral Treg generation and demonstrates the limited plasticity of Th1 populations.


Assuntos
Subpopulações de Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Células Th1/citologia , Transferência Adotiva , Animais , Diferenciação Celular/imunologia , Citometria de Fluxo , Fatores de Transcrição Forkhead , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia
17.
Blood ; 113(2): 389-95, 2009 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18815283

RESUMO

Imbalance of T-helper cell (Th) differentiation and subsequent cytokine dysregulation is implicated in inflammatory and autoimmune diseases. In particular, 2 cytokines produced by different Th cell populations, interferon-gamma (IFN-gamma) and interleukin-17 (IL-17), have been shown to play a critical role in autoimmunity. We have examined the roles of these cytokines in a mouse model of systemic autoimmunity resulting from the deletion of IL-2 in which autoimmune hemolytic anemia (AIHA) is a prominent feature. We demonstrate that, in IL-2-knockout (KO) BALB/c mice, elimination of the Th1 cytokine, IFN-gamma, delays the development of AIHA. Further, CD4(+) T cells from IL-2/IFN-gamma-KO mice produce elevated levels of IL-17 compared with wild-type (WT) and IL-2-KO, and these mice eventually develop intestinal inflammation. In contrast, elimination of the Th17 cytokine, IL-17, from IL-2-KO mice fails to suppress early acute AIHA development. These results suggest that in a systemic autoimmune disease with multiple manifestations, Th1 cells drive the early autoantibody response and IL-17-producing cells may be responsible for the more chronic tissue inflammation.


Assuntos
Anemia Hemolítica Autoimune/imunologia , Autoimunidade , Citocinas/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Doença Aguda , Anemia Hemolítica Autoimune/genética , Anemia Hemolítica Autoimune/metabolismo , Anemia Hemolítica Autoimune/patologia , Animais , Autoimunidade/genética , Doença Crônica , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Enteropatias/genética , Enteropatias/imunologia , Enteropatias/metabolismo , Enteropatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Células Th1/metabolismo , Células Th1/patologia
18.
Commun Biol ; 4(1): 561, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980972

RESUMO

Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.


Assuntos
Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Proteínas de Membrana/análise , Anticorpos Monoclonais/biossíntese , Anidrase Carbônica IX , Linhagem Celular , Simulação por Computador , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cadeias alfa de Integrinas , Proteínas de Membrana/imunologia , Biologia Sintética/métodos , Tetraspaninas
19.
Invest Ophthalmol Vis Sci ; 62(13): 15, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34665194

RESUMO

Purpose: Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFß activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Methods: Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. Results: USP10 overexpression increased α5ß1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5ß1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Conclusions: Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.


Assuntos
Córnea/metabolismo , Fibronectinas/metabolismo , Ubiquitina Tiolesterase/biossíntese , Adesão Celular , Membrana Celular/metabolismo , Células Cultivadas , Córnea/citologia , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Transdução de Sinais
20.
J Mol Biol ; 433(15): 167090, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34090922

RESUMO

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFß) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFß. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvß1, αvß3 and αvß5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Epitopos/metabolismo , Integrina alfaV/química , Neoplasias Pulmonares/metabolismo , Células A549 , Animais , Anticorpos Biespecíficos/química , Antineoplásicos Imunológicos/química , Células CHO , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Integrina alfaV/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Biblioteca de Peptídeos
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