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1.
EBioMedicine ; 44: 50-59, 2019 Jun.
Artigo em Francês | MEDLINE | ID: mdl-31097410

RESUMO

BACKGROUND: Clinical trials on multiple sclerosis with repeated injections of monoclonal antibodies depleting CD4+ T cells have not resulted in much success as a disease therapy. Here, we developed an immunotherapy for EAE in mice by combining a transient depletion of T cells together with the administration of neuron derived peptides. METHODS: EAE was induced in SJL and C57BL/6 mice, by proteolipid protein peptide PLP139-151 (pPLP) and myelin-oligodendrocyte glycoprotein MOG35-55 (pMOG) peptides, respectively. Anti-CD4 and anti-CD8 antibody were injected intraperitoneally before or after peptide immunization. EAE scores were evaluated and histology data from brain and spinal cord were analyzed. Splenocytes were isolated and CD4+, CD4+CD25- and CD4+CD25+ T cells were purified and cultured in the presence of either specific peptides or anti-CD3 antibody and proliferation of T cells as well as cytokines in supernatant were assessed. FINDINGS: This experimental treatment exhibited therapeutic effects on mice with established EAE in pPLP-susceptible SJL mice and pMOG-susceptible C57BL/6 mice. Mechanistically, we revealed that antibody-induced apoptotic T cells triggered macrophages to produce TGFß, and together with administered auto-antigenic peptides, generated antigen-specific Foxp3+ regulatory T cells (Treg cells) in vivo. INTERPRETATION: We successfully developed a specific immunotherapy to EAE by generating autoantigen-specific Treg cells. These findings have overcome the drawbacks of long and repeated depletion of CD4+ T cells, but also obtained long-term immune tolerance, which should have clinical implications for the development of a new effective therapy for multiple sclerosis. FUND: This research was supported by the Intramural Research Program of the NIH, NIDCR.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/imunologia , Autoantígenos/administração & dosagem , Encefalomielite Autoimune Experimental/imunologia , Peptídeos/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Biomarcadores/metabolismo , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Tolerância Imunológica , Fatores Imunológicos , Imunoterapia , Ativação Linfocitária , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Fagócitos/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Resultado do Tratamento
2.
J Pharmacol Sci ; 139(3): 193-200, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827890

RESUMO

Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H2O2-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1-8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30-1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury.


Assuntos
Apoptose/efeitos dos fármacos , Autoantígenos/administração & dosagem , Cardiotônicos/administração & dosagem , Colágeno Tipo IV/administração & dosagem , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Autoantígenos/química , Autoantígenos/farmacologia , Cardiotônicos/farmacologia , Linhagem Celular , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/complicações , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
3.
PLoS One ; 14(3): e0214379, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908554

RESUMO

Autoantigen-specific methods to prevent and treat Type 1 Diabetes (T1D) carry high hopes to permanently cure this disease, but have largely failed in clinical trials. One suggested approach to increase the efficacy of islet antigen-specific vaccination is to combine it with a modulator of the T cell response, with the goal of reducing effector differentiation and promoting regulatory T cells (Tregs). Here we asked if addition of antibodies that block the IL-7/IL-7Rα pathway altered the T cell response to islet antigen vaccination and prevented T1D in non-obese diabetic (NOD) mice. Anti-IL-7Rα monoclonal antibodies (mAbs) reduced the numbers of islet antigen-specific T cells generated after vaccination with islet peptides and alum. However, addition of anti-IL-7Rα antibodies to peptide/alum vaccination unexpectedly increased non-specific IFN-γ, IL-2 and IL-10 cytokine production and did not result in improved prevention of T1D onset. In a second approach, we used a conjugate vaccine to deliver islet autoantigens, using Keyhole Limpet Hemocyanin (KLH) as a carrier. Islet antigen-KLH vaccination led to a significant expansion of antigen-specific Tregs and delayed diabetes onset in NOD mice. These outcomes were not further improved by addition of anti-IL-7Rα antibodies. To the contrary, blocking IL-7Rα during vaccination led to non-specific cytokine production and reduced the efficacy of a KLH-conjugated vaccine to prevent T1D. Our study thus revealed that adding anti-IL-7Rα antibodies during autoantigen immunization did not improve the efficacy of such vaccinations to prevent T1D, despite altering some aspects of the T cell response in a potentially advantageous way. Further refinement of this approach will be required to separate the beneficial from the adverse effects of anti-IL-7Rα antibodies to treat autoimmune disease.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Autoantígenos/administração & dosagem , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Subunidade alfa de Receptor de Interleucina-7/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Autoantígenos/imunologia , Proliferação de Células , Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Imunização , Imunoterapia , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Vacinação
4.
Int J Pharm ; 562: 303-312, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30910633

RESUMO

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.


Assuntos
Autoantígenos/administração & dosagem , Ouro/administração & dosagem , Imunoterapia , Nanopartículas Metálicas/administração & dosagem , Peptídeos/administração & dosagem , Pele/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Humanos , Injeções Intradérmicas , Pessoa de Meia-Idade , Pele/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
5.
Mol Pharm ; 16(2): 607-617, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30615457

RESUMO

Contemporary approaches to treating autoimmune diseases like multiple sclerosis broadly modulate the immune system and leave patients susceptible to severe adverse effects. Antigen-specific immunotherapies (ASIT) offer a unique opportunity to selectively suppress autoreactive cell populations but have suffered from marginal efficacy even when employing traditional adjuvants to improve delivery. The development of immunologically active antigen delivery vehicles could potentially increase the clinical success of antigen-specific immunotherapies. An emulsion of the antioxidant tocopherol delivering an epitope of proteolipid protein autoantigen (PLP139-151) yielded significant efficacy in mice with experimental autoimmune encephalomyelitis (EAE). In vitro studies indicated tocopherol emulsions reduced oxidative stress in antigen-presenting cells. Ex vivo analysis revealed that tocopherol emulsions shifted cytokine responses in EAE splenocytes. In addition, IgG responses against PLP139-151 were increased in mice treated with tocopherol emulsions delivering the antigen, suggesting a possible skew in immunity. Overall, tocopherol emulsions provide a functional delivery vehicle for ASIT capable of ameliorating autoimmunity in a murine model.


Assuntos
Autoantígenos/uso terapêutico , Emulsões/química , Encefalomielite Autoimune Experimental/tratamento farmacológico , Tocoferóis/química , Tocoferóis/uso terapêutico , Animais , Autoantígenos/administração & dosagem , Citocinas/metabolismo , Feminino , Tolerância Imunológica/efeitos dos fármacos , Imunoterapia/métodos , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Baço/citologia
6.
Front Immunol ; 9: 2871, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30574145

RESUMO

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Fator Ativador de Células B/metabolismo , Tolerância Imunológica , Interleucina-33/imunologia , Animais , Autoantígenos/administração & dosagem , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Células Dendríticas , Modelos Animais de Doenças , Humanos , Imunoglobulina M/imunologia , Interleucina-33/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos , Cultura Primária de Células , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia
7.
Pharmazie ; 73(12): 715-720, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30522555

RESUMO

Tumstatin7 (CNYYSNS) is an antitumor peptide derived from the NC1 domain of Type IV collagen that has been associated with tumor angiogenesis. In this work, we generated a peptide composed of tumstatin7 fused to TAT, a cell-internalizing peptide consisting of 11 amino acids. Tumstatin7-TAT was internalized by cells and triggered cell death. The new peptide was more potent in inducing B16F10 melanoma cell apoptosis in vitro than the shorter tumstatin7. Whereas tumstatin7-TAT significantly reduced tumor cell viability, tumstatin7 showed only weak effects even at the highest treatment concentration applied. Both tumstatin7-TAT and tumstatin7 inhibited cell migration in an in vitro wound healing model, and the former was more effective than the latter in inhibiting tumor growth in vivo. Combining the cell-internalizing property of TAT with the tumor-specific property of tumstatin7 may provide a useful adjunct to tumor therapy.


Assuntos
Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Produtos do Gene tat/metabolismo , Melanoma Experimental/tratamento farmacológico , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/administração & dosagem , Autoantígenos/química , Movimento Celular/efeitos dos fármacos , Colágeno Tipo IV/administração & dosagem , Colágeno Tipo IV/química , Feminino , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Peptídeos/química , Cicatrização/efeitos dos fármacos
8.
Vaccine ; 36(52): 8008-8018, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30416020

RESUMO

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.


Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Imunoterapia/métodos , Insulina/imunologia , Precursores de Proteínas/imunologia , Animais , Autoantígenos/administração & dosagem , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Quimioterapia Combinada , Feminino , Insulina/genética , Interleucina-10/administração & dosagem , Interleucina-10/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Precursores de Proteínas/genética , Salmonella , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/imunologia
9.
Crit Rev Immunol ; 38(3): 207-231, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30004858

RESUMO

Oral tolerance can be defined as an inhibition of specific immune responsiveness to subsequent parenteral injections of proteins to which an individual or animal has been previously exposed via the oral route. Multiple mechanisms of tolerance are induced by oral-fed antigens, but induction of regulatory CD4 T-cells expressing the transcription factor Foxp3 and the membrane-bound TGF-ß stands out as the major players in oral tolerance. Oral antigen administration suppresses several animal models of autoimmune disease, including experimental autoimmune encephalomyelitis, uveitis, thyroiditis, myasthenia, arthritis, and diabetes, but also nonautoimmune inflammatory conditions such as asthma, atherosclerosis, graft rejection, allergy, and stroke. However, human trials have produced mixed results, and a great deal remains to be learned about the mechanisms of oral tolerance before it can be successfully applied to people. In this review, we highlight the cellular components involved in oral tolerance induction. A deep knowledge of these intricate cell interactions will pave the way for a successful application of antigen tolerance to treat autoimmune and nonautoimmune inflammatory diseases.


Assuntos
Doenças Autoimunes/terapia , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Administração Oral , Animais , Autoantígenos/administração & dosagem , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fator de Crescimento Transformador beta/metabolismo
10.
J Control Release ; 266: 156-165, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-28963036

RESUMO

Current therapies for autoimmune diseases focus on treating the symptoms rather than the underlying disease cause. A major setback in improving current therapeutics for autoimmunity is the lack of antigen specificity. Successful antigen-specific immunotherapy (ASIT) would allow for improved treatment of autoimmune diseases. In this work, dexamethasone was co-delivered with autoantigen (PLP) in vivo to create effective ASIT for the treatment of experimental autoimmune encephalomyelitis (EAE). Using an emulsion of incomplete Freund's adjuvant (IFA) as a co-delivery vehicle, it was discovered that the controlled release of autoantigen was important for the suppression of clinical disease symptoms. Analysis of the immune response via cytokines revealed that dexamethasone was important for shifting the immune response away from inflammation. Co-delivery of both autoantigen and dexamethasone increased B-cell populations and antibody production, signifying an increased humoral immune response. Overall, this data indicated that the co-delivery of PLP and dexamethasone with a water-in-oil emulsion is effective in treating a murine autoimmune model.


Assuntos
Anti-Inflamatórios/administração & dosagem , Autoantígenos/administração & dosagem , Dexametasona/administração & dosagem , Encefalomielite Autoimune Experimental/tratamento farmacológico , Adjuvante de Freund/administração & dosagem , Fatores Imunológicos/administração & dosagem , Lipídeos/administração & dosagem , Proteína Proteolipídica de Mielina/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Camundongos , Baço/citologia
11.
Nanomedicine (Lond) ; 12(11): 1231-1242, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28593827

RESUMO

AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.


Assuntos
Autoantígenos/administração & dosagem , Imunoterapia/métodos , Lipossomos/química , Esclerose Múltipla/terapia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Peptídeos/administração & dosagem , Fosfatidilserinas/química , Animais , Autoantígenos/imunologia , Autoantígenos/uso terapêutico , Feminino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/uso terapêutico , Peptídeos/imunologia , Peptídeos/uso terapêutico , Linfócitos T Reguladores/imunologia
12.
PLoS One ; 12(3): e0173176, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28257518

RESUMO

Although the role of T cells in autoimmunity has been explored for many years, the mechanisms leading to the initial priming of an autoimmune T cell response remain enigmatic. The 'hit and run' model suggests that self-antigens released upon cell death can provide the initial signal for a self-sustaining autoimmune response. Using a novel transgenic mouse model where we could induce the release of self-antigens via caspase-dependent apoptosis. We tracked the fate of CD8+ T cells specific for the self-antigen. Our studies demonstrated that antigens released from apoptotic cells were cross-presented by CD11c+ cells in the draining lymph node. This cross-presentation led to proliferation of self-antigen specific T cells, followed by a transient ability to produce IFN-γ, but did not lead to the development of autoimmune diabetes. Using this model we examined the consequences on T cell immunity when apoptosis was combined with dendritic cell maturation signals, an autoimmune susceptible genetic background, and the deletion of Tregs. The results of our study demonstrate that autoimmune diabetes cannot be initiated by the presentation of antigens released from apoptotic cells in vivo even in the presence of factors known to promote autoimmunity.


Assuntos
Autoantígenos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/imunologia , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Apoptose/genética , Autoantígenos/imunologia , Autoimunidade/genética , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Interferon gama/genética , Camundongos , Camundongos Transgênicos/imunologia
14.
Clin Cancer Res ; 23(10): 2478-2490, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-27965307

RESUMO

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478-90. ©2016 AACR.


Assuntos
Neoplasias Encefálicas/terapia , Neoplasias da Mama/terapia , Imunoterapia Adotiva , Receptor ErbB-2/imunologia , Antígeno gp100 de Melanoma/imunologia , Animais , Autoantígenos/administração & dosagem , Autoantígenos/imunologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/imunologia , Camundongos , Camundongos Transgênicos , Receptor ErbB-2/genética , Receptores de Antígenos de Linfócitos T/administração & dosagem , Receptores de Antígenos de Linfócitos T/imunologia , Indução de Remissão , Vírus Vaccinia/genética , Vírus Vaccinia/imunologia , Antígeno gp100 de Melanoma/genética
15.
J Neuroimmune Pharmacol ; 11(4): 749-762, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27518777

RESUMO

Parasite proteins containing repeats are essential invasion ligands, important for their ability to evade the host immune system and to induce immunosuppression. Here, the intrinsic suppressive potential of repetitive structures within parasite proteins was exploited to induce immunomodulation in order to establish self-tolerance in an animal model of autoimmune neurological disease. We tested the tolerogenic potential of fusion proteins containing repeat sequences of parasites linked to self-antigens. The fusion constructs consist of a recombinant protein containing repeat sequences derived from the S-antigen protein (SAg) of Plasmodium falciparum linked to a CD4 T cell epitope of myelin. They were tested for their efficacy to control the development of experimental autoimmune encephalomyelitis (EAE), In addition, we used the DO11.10 transgenic mouse model to study the immune mechanisms involved in tolerance induced by SAg fusion proteins. We found that repeated sequences of P. falciparum SAg protein linked to self-epitopes markedly protected mice from EAE. These fusion constructs were powerful tolerizing agents not only in a preventive setting but also in the treatment of ongoing disease. The tolerogenic effect was shown to be antigen-specific and strongly dependent on the physical linkage of the T cell epitope to the parasite structure and on the action of anti-inflammatory cytokines like IL-10 and TGF-ß. Other mechanisms include down-regulation of TNF-α accompanied by increased numbers of FoxP3+ cells. This study describes the use of repetitive structures from parasites linked to defined T cell epitopes as an effective method to induce antigen-specific tolerance with potential applicability for the treatment and prevention of autoimmune diseases.


Assuntos
Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Plasmodium falciparum/imunologia , Sequências Repetitivas de Ácido Nucleico/imunologia , Sequência de Aminoácidos , Animais , Autoantígenos/administração & dosagem , Autoantígenos/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Encefalomielite Autoimune Experimental/genética , Feminino , Imunização/métodos , Imunomodulação/efeitos dos fármacos , Imunomodulação/genética , Imunomodulação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parasitos/genética , Parasitos/imunologia , Plasmodium falciparum/genética , Sequências Repetitivas de Ácido Nucleico/genética
17.
Sci Rep ; 6: 26309, 2016 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-27199164

RESUMO

Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 µm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.


Assuntos
Asma/fisiopatologia , Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Pulmão/patologia , Remodelação Vascular/efeitos dos fármacos , Resistência das Vias Respiratórias/efeitos dos fármacos , Alérgenos/administração & dosagem , Animais , Asma/imunologia , Autoantígenos/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Doença Crônica , Colágeno Tipo IV/administração & dosagem , Dermatophagoides pteronyssinus/imunologia , Feminino , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Músculo Liso/metabolismo , Carneiro Doméstico , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
PLoS One ; 11(1): e0147260, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26783749

RESUMO

Induction of mucosal tolerance by oral administration of protein antigens is a potential therapeutic strategy for preventing and treating type 1 diabetes (T1D); however, the requirement for a large dosage of protein limits clinical applications because of the low efficacy. In this study, we generated a fusion protein CTB-Ins-GAD composed of CTB (cholera toxin B subunit), insulin, and three copies of GAD65 peptide 531-545, which were efficiently produced in silkworm pupae, to evaluate its protective effect against T1D. We demonstrate that oral administration of CTB-Ins-GAD suppressed T1D by up to 78%, which is much more effective than GAD65 single-antigen treatment. Strikingly, CTB-Ins-GAD enhance insulin- and GAD65-specific Th2-like immune responses, which repairs the Th1/Th2 imbalance and increases the number of CD4(+)CD25(+)Foxp3(+) T cell and suppresses insulin- and GAD65-reactive spleen T lymphocyte proliferation and migration. Our results strongly suggest that the combined dual antigens promote the induction of oral tolerance, thus providing an effective and economic immunotherapy against T1D in combination with a silkworm bioreactor.


Assuntos
Autoantígenos/administração & dosagem , Bombyx/enzimologia , Diabetes Mellitus Tipo 1/prevenção & controle , Glutamato Descarboxilase/administração & dosagem , Insulina/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/imunologia , Administração Oral , Transferência Adotiva , Animais , Autoantígenos/imunologia , Western Blotting , Diferenciação Celular , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/imunologia , Feminino , Glutamato Descarboxilase/imunologia , Incidência , Insulina/imunologia , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese
19.
J Control Release ; 223: 178-187, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26739548

RESUMO

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional "wet" intradermal (ID) administration, "dry" peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases.


Assuntos
Autoantígenos/administração & dosagem , Cromogranina A/administração & dosagem , Diabetes Mellitus Tipo 1/terapia , Imunoterapia/métodos , Fragmentos de Peptídeos/administração & dosagem , Administração Tópica , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Apresentação do Antígeno/efeitos dos fármacos , Autoantígenos/uso terapêutico , Betametasona/administração & dosagem , Betametasona/uso terapêutico , Cromogranina A/uso terapêutico , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Camundongos Transgênicos , Microinjeções , Pessoa de Meia-Idade , Agulhas , Fragmentos de Peptídeos/uso terapêutico , Pele/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto Jovem
20.
Technol Cancer Res Treat ; 15(3): 498-508, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-25969440

RESUMO

Tumstatin (Tum) is a powerful angiostatin that inhibits proliferation and induces apoptosis of tumorous vascular endothelial cells. A nonpathogenic and anaerobic bacterium, Bifidobacterium longum (BL), selectively localizes to and proliferates in the hypoxia location within solid tumor. The aims of this study were to develop a novel delivery system for Tum using engineered Bifidobacterium and to investigate the inhibitory effect of Tum on tumor in mice. A vector that enabled the expression of Tum under the control of the pBBADs promoter of BL was constructed and transformed into BL NCC2705 by electroporation. The mouse colon carcinoma cells CT26 (1 × 10(7)/mL) were subcutaneously inserted in the left armpit of BALB/c mice. The tumor-bearing mice were treated with Tum-transformed BL, and green fluorescent protein (GFP)-transformed BL was used as a negative control. The microvessel density (MVD) in the transplanted tumor was determined, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling was used to detect apoptosis of vascular endothelial cells in transplanted tumor. The in vitro expression of Tum was examined in BL after l-arabinose induction. Bifidobacterium longum with pBBAD-Tum (BL-Tum) showed significant antitumor effect in tumor-bearing mice. The weight, volume, growth, and MVD, as well as the percentage of apoptotic vascular endothelial cells of transplanted tumors in the tumor-bearing mice treated with Tum-transformed BL were all significantly lower than those in the GFP negative control group. Intragastric administration, injection in tumor and vena caudalis injection of Tum-transformed BL exerted marked antitumor effects in tumor-bearing mice. This is the first demonstration of the utilization of Tum-transformed BL as a specific gene delivery system for treating tumor.


Assuntos
Autoantígenos/administração & dosagem , Colágeno Tipo IV/administração & dosagem , Terapia Genética/métodos , Neoplasias Experimentais/patologia , Animais , Autoantígenos/genética , Bifidobacterium longum , Colágeno Tipo IV/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C
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