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1.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32630037

RESUMO

Preparations of comfrey (Symphytum officinale L.) roots are used topically to reduce inflammation. Comfrey anti-inflammatory and analgesic properties have been proven in clinical studies. However, the bioactive compounds associated with these therapeutic activities are yet to be identified. An LC-ESI-Orbitrap-MSn metabolite profile of a hydroalcoholic extract of comfrey root guided the identification of 20 compounds, including a new arylnaphthalene lignan bearing a rare δ-lactone ring, named comfreyn A. Its structure was determined using extensive 2D NMR and ESI-MS experiments. Additionally, the occurrence of malaxinic acid, caffeic acid ethyl ester, along with the lignans ternifoliuslignan D, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl) -naphthalene, globoidnan A and B, and rabdosiin was reported in S. officinale for the first time. These results helped to redefine the metabolite profile of this medicinal plant. Finally, caffeic acid ethyl ester and comfreyn A were found to significantly inhibit E-selectin expression in IL-1ß stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 µM, respectively.


Assuntos
Confrei/química , Confrei/metabolismo , Anti-Inflamatórios/análise , Cromatografia Líquida , Células Endoteliais da Veia Umbilical Humana , Humanos , Estrutura Molecular , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Plantas Medicinais/química , Plantas Medicinais/metabolismo , Espectrometria de Massas por Ionização por Electrospray
2.
Cell Rep Methods ; 4(4): 100728, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38492569

RESUMO

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.


Assuntos
Receptores ErbB , Receptores de Antígenos Quiméricos , Linfócitos T , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia Adotiva/métodos , Animais , Neoplasias/imunologia , Neoplasias/terapia , Linhagem Celular Tumoral , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos
3.
Sci Rep ; 13(1): 23024, 2023 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-38155191

RESUMO

The majority of approved CAR T cell products are based on the FMC63-scFv directed against CD19. Surprisingly, although antigen binding affinity is a major determinant for CAR function, the affinity of the benchmark FMC63-scFv has not been unambiguously determined. That is, a wide range of affinities have been reported in literature, differing by more than 100-fold. Using a range of techniques, we demonstrate that suboptimal experimental designs can cause artefacts that lead to over- or underestimation of the affinity. To minimize these artefacts, we performed SPR with strictly monomeric and correctly folded soluble CD19, yielding an FMC63-scFv affinity of 2-6 nM. Together, apart from analyzing the FMC63-scFv affinity under optimized conditions, we also provide potential explanations for the wide range of published affinities. We expect that this study will be highly valuable for interpretations of CAR affinity-function relationships, as well as for the design of future CAR T cell generations.


Assuntos
Imunoterapia Adotiva , Linfócitos T , Imunoterapia Adotiva/métodos , Antígenos CD19
4.
ACS Synth Biol ; 10(5): 1184-1198, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33843201

RESUMO

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.


Assuntos
Substituição de Aminoácidos , Antígenos CD19/genética , Evolução Molecular Direcionada/métodos , Imunoterapia Adotiva/métodos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Aminoácidos/genética , Anticorpos Monoclonais/imunologia , Antígenos CD19/química , Antígenos CD19/imunologia , Células HEK293 , Humanos , Linfoma Difuso de Grandes Células B/sangue , Proteínas Mutantes , Mutação , Domínios Proteicos/imunologia , Dobramento de Proteína , Estabilidade Proteica , Receptores de Antígenos Quiméricos/genética
5.
J Biotechnol ; 331: 1-13, 2021 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-33689865

RESUMO

Despite the essential role secretory IgAs play in the defense against pathogenic invasion and the proposed value of recombinant secretory IgAs as novel therapeutics, currently there are no IgA-based therapies in clinics. Secretory IgAs are complex molecules and the major bottleneck limiting their therapeutic potential is a reliable recombinant production system. In this report, we addressed this issue and established a fast and robust production method for secretory IgAs in CHO-K1 cells using BAC-based expression vectors. As a proof of principle, we produced IgAs against Clostridium difficile toxins TcdA and TcdB. Recombinant secretory IgAs produced using our expression system showed comparable titers to IgGs, widely used as therapeutic biologicals. Importantly, secretory IgAs produced using our method were functional and could efficiently neutralize Clostridium difficile toxins TcdA and TcdB. These results show that recombinant secretory IgAs can be efficiently produced, thus opening the possibility to use them as therapeutic agents in clinics.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Animais , Proteínas de Bactérias , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Cricetinae , Enterotoxinas/genética , Imunoglobulina A Secretora
6.
Front Pharmacol ; 10: 289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105555

RESUMO

Symphytum officinale, commonly known as comfrey, constitutes a traditional medicinal plant with a long-standing therapeutic history, and preparations thereof have been widely used for the treatment of painful muscle and joint complaints, wound and bone healing, and inflammation. Today, its topical use is based on its analgesic and anti-inflammatory effects, which have been substantiated by modern clinical trials. However, the molecular basis of its action remained elusive. Here, we show that a hydroalcoholic extract of comfrey root impairs the development of a pro-inflammatory scenario in primary human endothelial cells in a dose-dependent manner. The extract, and especially its mucilage-depleted fraction, impair the interleukin-1 (IL-1) induced expression of pro-inflammatory markers including E-selectin, VCAM1, ICAM1, and COX-2. Both preparations inhibit the activation of NF-κB, a transcription factor of central importance for the expression of these and other pro-inflammatory genes. Furthermore, our biochemical studies provide evidence that comfrey inhibits NF-κB signaling at two stages: it dampens not only the activation of IKK1/2 and the subsequent IκBα degradation, but also interferes with NF-κB p65 nucleo-cytoplasmatic shuttling and transactivation. These results provide a first mechanistic insight into the mode of action of a century-old popular herbal medicine.

7.
Vascul Pharmacol ; 90: 44-50, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28192257

RESUMO

AIMS: Levosimendan is an inodilator for the treatment of acute decompensated heart failure (HF). Data from clinical studies suggest that levosimendan is particularly effective in HF due to myocardial infarction. After acute revascularization, no reflow-phenomenon is a common complication that may lead to pump failure and cardiogenic shock. Our aim was to examine whether levosimendan interferes with the pro-thrombotic phenotype of activated endothelial cells in vitro. METHODS: Human heart microvascular endothelial cells (HHMEC) and human umbilical vein endothelial cells (HUVEC) were treated with interleukin-1ß (IL-1ß) (200U/mL) or thrombin (5U/mL) and co-treated with or without levosimendan (0.1-10µM) for 2-24h. In addition, flow experiments were performed. Effects on plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) expression and activity were measured by rt-PCR, specific ELISA and flow cytometry. RESULTS: Treatment with IL-1ß or thrombin significantly increased the expression of PAI-1 and TF in endothelial cells. Co-treatment with levosimendan strongly attenuated the effects of IL-1ß and thrombin on PAI-1 and TF mRNA by up to 50% and 45%, in a dose- and time-dependent manner. Similar results were obtained under flow conditions. Furthermore, co-treatment with levosimendan dampened the antigen production of PAI-1 and the surface expression of TF by 35% and 45%, respectively. Additionally, levosimendan diminished both TF and PAI-1 activity. CONCLUSION: Levosimendan down-regulates the expression of the pro-thrombotic and anti-fibrinolytic biomolecules TF and PAI-1 in activated human endothelial cells. Our findings may, at least in part, explain some of the beneficial effects of levosimendan after myocardial reperfusion.


Assuntos
Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Hidrazonas/farmacologia , Piridazinas/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1beta/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Simendana , Trombina/farmacologia , Tromboplastina/metabolismo , Fatores de Tempo
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