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1.
Front Immunol ; 15: 1360063, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38558809

RESUMO

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Riboswitch , Camundongos , Humanos , Animais , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Terapia Genética , Interleucina-12/genética , Interleucina-12/metabolismo , Tetraciclina/farmacologia
2.
NPJ Parkinsons Dis ; 9(1): 91, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37322068

RESUMO

Prion-like transmission of pathology in α-synucleinopathies like Parkinson's disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier.

3.
Immunohorizons ; 7(3): 200-212, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36928178

RESUMO

Regulatory T cells (Treg) play a critical role in controlling immune responses in diseases such as cancer or autoimmunity. Activated Treg express the membrane protein GARP (LRRC32) in complex with the latent form of the immunosuppressive cytokine TGF-ß (L-TGF-ß). In this study, we confirmed that active TGF-ß was generated from its latent form in an integrin-dependent manner and induced TGF-ß receptor signaling in activated human Treg. We studied a series of Abs targeting the L-TGF-ß/GARP complex with distinct binding modes. We found that TGF-ß receptor signaling could be inhibited by anti-TGF-ß and by some, but not all, Abs against the L-TGF-ß/GARP complex. Cryogenic electron microscopy structures of three L-TGF-ß/GARP complex-targeting Abs revealed their distinct epitopes and allowed us to elucidate how they achieve blockade of TGF-ß activation. Three different modes of action were identified, including a novel unusual mechanism of a GARP-binding Ab. However, blockade of GARP or TGF-ß by Abs did not influence the suppressive activity of human Treg in vitro. We were also not able to confirm a prominent role of GARP in other functions of human Treg, such as FOXP3 induction and Treg stability. These data show that the GARP/TGF-ß axis can be targeted pharmacologically in different ways, but further studies are necessary to understand its complexity and to unleash its therapeutic potential.


Assuntos
Anticorpos , Linfócitos T Reguladores , Fator de Crescimento Transformador beta , Humanos , Proteínas de Membrana/imunologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Anticorpos/farmacologia
4.
J Cell Biol ; 222(2)2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36459066

RESUMO

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.


Assuntos
Apoptose , Antígeno CD47 , Macrófagos , Fenótipo Secretor Associado à Senescência , Animais , Humanos , Camundongos , Aminoaciltransferases/metabolismo , Antígeno CD24/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Macrófagos/citologia , Regulação para Cima
5.
Biomedicines ; 10(9)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36140247

RESUMO

One important prerequisite for developing a therapeutic monoclonal antibody is to evaluate its in vivo efficacy. We tested the therapeutic potential of an anti-CD96 antibody alone or in combination with an anti-PD-1 antibody in a mouse colon cancer model. Early anti-PD-1 treatment significantly decreased tumor growth and the combination with anti-CD96 further increased the therapeutic benefit, while anti-CD96 treatment alone had no effect. In late therapeutic settings, the treatment combination resulted in enhanced CD8+ T cell infiltration of tumors and an increased CD8/Treg ratio. Measured anti-PD-1 concentrations were as expected in animals treated with anti-PD-1 alone, but lower at later time points in animals receiving combination treatment. Moreover, anti-CD96 concentrations dropped dramatically after 10 days and were undetectable thereafter in most animals due to the occurrence of anti-drug antibodies that were increasing antibody clearance. Comparison of the anti-PD-1 concentrations with tumor growth showed that higher antibody concentrations in plasma correlated with better therapeutic efficacy. The therapeutic effect of anti-CD96 treatment could not be evaluated, because plasma concentrations were too low. Our findings strongly support the notion of measuring both plasma concentration and anti-drug antibody formation throughout in vivo studies, in order to interpret pharmacodynamic data correctly.

6.
J Ocul Pharmacol Ther ; 33(2): 115-122, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28055308

RESUMO

PURPOSE: The ocular half-life of intravitreally (IVT) injected drugs is of major relevance for the suitability of a drug intended for chronic intraocular treatment, as the half-life determines the dosing frequency. Thus, half-life extension principles are very attractive as they can reduce the IVT dosing frequency. In this study, we investigated the ocular pharmacokinetics (PK) of the IVT injected Nanobody® BI-X and whether the noncovalent binding of BI-X to vitreous albumin could increase its ocular half-life. METHODS: Wistar rats were dosed IVT with 3 µg BI-X alone or coadministered with human serum albumin (HSA), and the ocular exposure was measured in a pilot experiment using whole eye homogenates. New Zealand White rabbits received IVT injections of 500 µg BI-X alone or coadministration with HSA. Concentrations of BI-X were determined in aqueous humor, vitreous body and plasma and pharmacokinetic parameters were calculated. RESULTS: Ocular concentrations of BI-X in rats were about 10- or 3-fold higher at 24 and 72 h, respectively, when dosed with HSA. In rabbits, coadministration with albumin led to an about 3-fold increased vitreous half-life and an about 5-fold higher exposure in vitreous humor. CONCLUSION: As small amounts of albumin are present in the vitreous body of healthy human eyes and the albumin concentration is even increased under disease conditions like diabetic retinopathy, high affinity binding to albumin may be a promising strategy to extend the half-life of IVT injected drugs, allowing for longer dosing intervals.


Assuntos
Humor Aquoso/metabolismo , Albumina Sérica/farmacocinética , Anticorpos de Domínio Único/metabolismo , Corpo Vítreo/metabolismo , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Meia-Vida , Humanos , Injeções Intravítreas , Masculino , Coelhos , Ratos , Ratos Wistar , Albumina Sérica/administração & dosagem , Albumina Sérica/química , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/química
7.
Biopreserv Biobank ; 9(3): 265-271, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21977242

RESUMO

There is a rising need for biomaterial in dermatological research with regard to both quality and quantity. Research biobanks as organized collections of biological material with associated personal and clinical data are of increasing importance. Besides technological/methodological and legal aspects, the willingness to donate samples by patients and healthy volunteers is a key success factor. To analyze the theoretical willingness to donate blood and skin samples, we developed and distributed a questionnaire. Six hundred nineteen questionnaires were returned and analyzed. The willingness to donate samples of blood (82.5%) and skin (58.7%) is high among the population analyzed and seems to be largely independent of any expense allowance. People working in the healthcare system, dermatological patients, and higher qualified individuals seem to be in particular willing to donate material. An adequate patient insurance as well as an extensive education about risks and benefits is requested. In summary, there is a high willingness to donate biological samples for dermatological research. This theoretical awareness fits well with our own experiences in establishing such a biobank.

8.
Pharmacol Ther ; 130(3): 371-84, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21420429

RESUMO

The mammalian transient receptor potential (TRP) superfamily of cation channels is divided into six subfamilies based on sequence homology TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin) and TRPML (mucolipin). The expression of these channels is especially abundant in sensory nerves, and there is increasing evidence demonstrating their existence in a broad range of cell types which are thought to play a key role in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). These ion channels can be activated by a diverse range of chemical and physical stimuli. Physical stimuli include temperature, membrane potential changes and osmotic stress, and some of the more well known chemical stimuli include capsaicin (TRPV1), menthol (TRPM8) and acrolein (TRPA1). There is increasing evidence in this rapidly moving field to suggest that selective blockers of these channels may represent attractive novel strategies to treat characteristic features of respiratory diseases such as asthma and COPD. This review focuses on summarising the evidence that modulation of selected TRP channels may have beneficial effects at targeting key features of these respiratory diseases including airways inflammation, airways hyper-reactivity, mucus secretion and cough.


Assuntos
Sistemas de Liberação de Medicamentos/tendências , Pneumopatias/tratamento farmacológico , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Capsaicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Humanos , Pneumopatias/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia
9.
J Invest Dermatol ; 127(4): 835-45, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17235328

RESUMO

A mixture of different fumaric acid esters (FAE) is established for systemic therapy of psoriasis, a frequent inflammatory skin disease. The main active compound of FAE, however, has not been identified so far, and the mechanisms of activity are only partially understood. We analyzed the impact of FAE on in vitro immune function and aimed to gain knowledge about the mode of action. Dimethylfumarate (DMF) and diethylfumarate (DEF), but not fumaric acid, methylhydrogenfumarate and ethylhydrogenfumarate, exhibited potent depression of inflammatory cytokine secretion (e.g., tumor necrosis factoralpha, IL-12, and IFNgamma) in activated human peripheral blood mononuclear cells. Moreover, solely DMF and DEF inhibited alloreactive T-cell proliferation in mixed leukocyte reaction. Interestingly, these immunosuppressive effects were accompanied by the strong induction of the anti-inflammatory stress protein heme oxygenase 1 (HO-1). Supplementation with exogenous glutathione (GSH), which is known to bind DMF, prevented both HO-1 induction as well as the anti-inflammatory effects of DMF. Moreover, inhibition of HO-1 activity restored the diminished IL-12 and IFNgamma production after FAE treatment. These results suggest that DMF acts as active compound within the FAE mixture and at least partially mediates its immunomodulatory activity by the induction of the anti-inflammatory stress protein HO-1 ascribed to the functional depletion of reduced GSH.


Assuntos
Fumaratos/farmacologia , Glutationa/antagonistas & inibidores , Heme Oxigenase-1/biossíntese , Imunossupressores/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/antagonistas & inibidores , Fumarato de Dimetilo , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Fumaratos/administração & dosagem , Glutationa/análogos & derivados , Glutationa/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Humanos , Imunossupressores/administração & dosagem , Mediadores da Inflamação/antagonistas & inibidores , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-12/metabolismo , Linfócitos/citologia , RNA Interferente Pequeno/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
10.
Expert Opin Drug Discov ; 1(1): 53-68, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23506032

RESUMO

Inflammatory skin diseases have a high prevalence in Western countries and pharmaceutical companies spend increasing amounts of money to develop drugs for these disorders. However, their complex pathophysiology is only partially reflected in classical rodent models, limiting their predictivity, and new compounds frequently fail in clinical trials. Therefore, there is an urgent need for more predictive and reliable animal models. Humanised mouse models seem to combine the convenience of small animal models with good correlation to the situation in patients and, thus, promise to be a valuable technique for translational medicine. Here, the authors summarise how mice can be humanised and which humanised models for inflammatory skin diseases exist. Application of these models in drug discovery and their advantages and limitations are discussed.

11.
Cancer Immunol Immunother ; 54(11): 1127-36, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15889255

RESUMO

Cancer development relies on a variety of mechanisms that facilitate tumor growth despite the presence of a functioning immune system. Understanding these mechanisms may foster novel therapeutic approaches for oncology and organ transplantation. By expression of the apoptosis-inducing protein CD95L (FasL, APO-1L, CD178), tumors may eliminate tumor-infiltrating lymphocytes and suppress anti-tumor immune responses, a phenomenon called "tumor counterattack". On the one hand, preliminary evidence of tumor counterattack in human tumors exists, and CD95L expression can prevent T-cell responses in vitro. On the other hand, CD95L-expressing tumors are rapidly rejected and induce inflammation in mice. Here, we summarize and discuss the consequences of CD95L expression of tumor cells and its contribution to immune escape.


Assuntos
Glicoproteínas de Membrana/fisiologia , Neoplasias/imunologia , Evasão Tumoral/imunologia , Apoptose , Proteína Ligante Fas , Humanos , Inflamação/etiologia , Glicoproteínas de Membrana/análise , Linfócitos T/imunologia , Receptor fas/fisiologia
12.
Int J Cancer ; 113(1): 78-87, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15386427

RESUMO

Many tumors express CD95L (CD178, FasL, APO-1L) and may thus kill tumor-infiltrating lymphocytes, a phenomenon called tumor counterattack. However, presently it is not clear whether tumor counterattack is a relevant immune escape mechanism. To characterize the effect of CD95L expression of tumor cells on tumor-specific T cells, we established an in vitro system with TCR tg T cells and a model tumor antigen. Preactivated antitumor T cells were able to kill CD95L(-) and CD95L(+) tumor cells. CD95L(+) tumor cells killed activated T cells in vitro and inhibited the expansion of cytotoxic antitumor T cells in mixed lymphocyte tumor reactions. In vivo CD95L expression led to delayed tumor growth or complete tumor rejection. Neutrophils were not responsible for the delayed growth of the CD95L(+) tumors tested. In mice with neutrophils deficient for important cytotoxicity mechanisms (p47phox(-/-) or iNOS(-/-) mice), CD95L(+) tumors grew similarly as in wild-type mice. Incidence and growth rate of CD95L(+) tumors in mice injected with a neutrophil-depleting or an isotype control antibody was the same. In CD95-deficient lpr mice, tumor growth was not altered as compared to wild-type mice. Taken together, CD95L mediated tumor counterattack in vitro, but led to neutrophil-independent tumor rejection in vivo.


Assuntos
Citotoxicidade Imunológica , Glicoproteínas de Membrana/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Proteína Ligante Fas , Feminino , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , NADPH Oxidases , Neutrófilos/metabolismo , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase Tipo II , Fosfoproteínas/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Trends Pharmacol Sci ; 25(10): 543-9, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15380939

RESUMO

Differences between humans and mice often hamper the transfer of promising results from the bench to the clinic. For ethical reasons, research that involves patients is limited, and so there is an urgent need for models that mimic the human situation as closely as possible. In recent years, there has been considerable progress in generating humanized mouse models, and their application to drug discovery has proved fruitful. So, how can mice be humanized, and how can humanized mice be employed in immunology research and drug discovery? In this article, we answer these questions, focusing on T-cell-mediated skin diseases as an example.


Assuntos
Dermatite/tratamento farmacológico , Dermatite/imunologia , Modelos Animais de Doenças , Camundongos/imunologia , Projetos de Pesquisa , Animais , Humanos , Especificidade da Espécie
15.
Eur J Immunol ; 33(10): 2811-21, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14515265

RESUMO

Many tumors express the death ligand CD95L (CD178, APO-1L, FasL) and can kill activated T cells in vitro. This may enable the tumor cells to suppress anti-tumor immune responses, a phenomenon called "tumor counterattack". Preliminary evidence of tumor counterattack in human tumors exists. However, CD95L-expressing tumors are rapidly rejected in mice. In order to clarify this controversial situation we investigated whether the level or the time point of CD95L expression might be critical factors determining tumor counterattack versus tumor rejection. We generated CD95-resistant tumor cell lines expressing different levels of CD95L (LKC-CD95L). In nude mice the CD95L expression level had no influence on the growth of the CD95L(+) tumors. In contrast, a CD95L(-) control tumor cell line (LKC) grew much faster. In addition, we generated a CD95-resistant cell line in which CD95L was induced via the tet system (LKCR-tetCD95L). Induction of CD95Lin established tumors in nude and NOD/SCID mice led to rapid rejection of the tumors. Induction of lower CD95L expression levels delayed tumor rejection only marginally. These results demonstrate that rejection of CD95L-expressing tumors in mice is not a result of overexpression and does not depend on the presence of CD95L at the onset of tumor progression.


Assuntos
Glicoproteínas de Membrana/fisiologia , Transplante de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Animais , Apoptose , Citotoxicidade Imunológica , Proteína Ligante Fas , Feminino , Rejeição de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologia , Células Tumorais Cultivadas
16.
J Leukoc Biol ; 71(6): 907-20, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12050175

RESUMO

Interactions between the immune system and malignant cells play an important role in tumorigenesis. Failure of the immune system to detect and reject transformed cells may lead to cancer development. Tumors use multiple mechanisms to escape from immune-mediated rejection. Many of these mechanisms are now known on a cellular and molecular level. Despite this knowledge, cancer immunotherapy is still not an established treatment in the clinic. This review discusses the immune escape mechanisms used by tumors with an emphasis on mechanisms related to apoptosis.


Assuntos
Apoptose/fisiologia , Sistema Imunitário/imunologia , Neoplasias/imunologia , Animais , Humanos , Imunoterapia/métodos , Modelos Biológicos , Neoplasias/patologia
17.
Nat Rev Cancer ; 2(4): 277-88, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12001989

RESUMO

Every cell in a multicellular organism has the potential to die by apoptosis, but tumour cells often have faulty apoptotic pathways. These defects not only increase tumour mass, but also render the tumour resistant to therapy. So, what are the molecular mechanisms of tumour resistance to apoptosis and how can we use this knowledge to resensitize tumour cells to cancer therapy?


Assuntos
Apoptose , Neoplasias/patologia , Animais , Regulação para Baixo , Humanos , Ligantes , Mitocôndrias/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
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