RESUMO
Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.
Assuntos
Indutores da Angiogênese/administração & dosagem , Sistemas de Liberação de Medicamentos , Proteína Forkhead Box M1/administração & dosagem , Fatores de Transcrição Forkhead/administração & dosagem , Hiperóxia/tratamento farmacológico , Nanopartículas , Alvéolos Pulmonares/efeitos dos fármacos , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/uso terapêutico , Animais , Animais Recém-Nascidos , Western Blotting , Feminino , Citometria de Fluxo , Proteína Forkhead Box M1/farmacologia , Proteína Forkhead Box M1/uso terapêutico , Fatores de Transcrição Forkhead/farmacologia , Fatores de Transcrição Forkhead/uso terapêutico , Hiperóxia/patologia , Hiperóxia/fisiopatologia , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do TratamentoRESUMO
BACKGROUND: With almost 47 million individuals worldwide suffering from some aspect of dementia, it is clear that cognitive loss impacts a significant proportion of the global population. Unfortunately, definitive treatments to resolve or prevent the onset of cognitive loss are limited. In most cases such care is currently non-existent prompting the need for novel treatment strategies. METHODS: Mammalian forkhead transcription factors of the O class (FoxO) are one such avenue of investigation that offer an exciting potential to bring new treatments forward for disorders that involve cognitive loss. Here we examine the background, structure, expression, and function of FoxO transcription factors and their role in cognitive loss, programmed cell death in the nervous system with apoptosis and autophagy, and areas to target FoxOs for dementia and specific disorders such as Alzheimer's disease. RESULTS: FoxO proteins work in concert with a number of other cell survival pathways that involve growth factors, such as erythropoietin and neurotrophins, silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), Wnt1 inducible signaling pathway protein 1 (WISP1), Wnt signaling, and cancer-related pathways. FoxO transcription factors oversee proinflammatory pathways, affect nervous system amyloid (Aß) production and toxicity, lead to mitochondrial dysfunction, foster neuronal apoptotic cell death, and accelerate the progression of degenerative disease. However, under some scenarios such as those involving autophagy, FoxOs also can offer protection in the nervous system and reduce toxic intracellular protein accumulations and potentially limit Aß toxicity. CONCLUSION: Given the ability of FoxOs to not only promote apoptotic cell death in the nervous system, but also through the induction of autophagy offer protection against degenerative disease that can lead to dementia, a fine balance in the activity of FoxOs may be required to target cognitive loss in individuals. Future work should yield exciting new prospects for FoxO proteins as new targets to treat the onset and progression of cognitive loss and dementia.
Assuntos
Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismo , Fatores de Transcrição Forkhead/administração & dosagem , Fatores de Transcrição Forkhead/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , HumanosRESUMO
The existence of a developed network of suppressory factors and cells against an immune response in different cancers has been proven; regulatory T cells are a typical issue. Therefore their depletion, elimination, or suppression has been assessed in different research studies that were not entirely successful. By applying an improved vaccine against regulatory T cells, we have evaluated the B cell response elicited by the vaccine in an experimental design. A previously described DNA vaccine and recombinant protein of Foxp3-Fc fusion were produced and used in the vaccination regimen. DNA construct and respective protein were injected into C57BL/6 mice. After 2 weeks, serum levels of IgG antibody and its subtypes against Foxp3 were investigated by ELISA. To produce recombinant Foxp3 for ELISA antigen coating, pET24a-Foxp3 vector was transformed into Escherichia coli strain BL21 as host cells. Afterward, protein was expressed and then purified using Ni-NTA agarose. SDS-PAGE and Western blot analysis were carried out to confirm protein expression. The expression analysis of Foxp3 was confirmed by SDS-PAGE followed by Western blot analysis. FOXP3-Fc DNA vaccine/fusion protein vaccination regimen could induce T helper-dependent humoral responses. Due to the effectiveness of Foxp3-Fc(IgG) in inducing humoral responses, it would be expected to be useful in developing vaccines in tumor therapies for the removal of regulatory T cells as a strategy for increasing the efficiency of other means of immunotherapy.
Assuntos
Vacinas Anticâncer/administração & dosagem , Fatores de Transcrição Forkhead/administração & dosagem , Imunidade Humoral/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacos , Vacinas de DNA/administração & dosagem , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/imunologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/biossíntese , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Vacinação , Vacinas de DNA/biossíntese , Vacinas de DNA/imunologiaRESUMO
Foxp3 is the master transcription factor for T regulatory (Treg) cell differentiation and function. This study aimed to test the therapeutic potential of cell penetrating recombinant Foxp3 protein in arthritis. Recombinant Foxp3 protein was fused to a cell penetrating polyarginine (Foxp3-11R) tag to facilitate intracellular transduction. In vitro Foxp3-11R treated CD4(+) T cells showed a 50% increase in suppressive function compared with control protein treated cells. Severity of arthritis in Foxp3-11R treated mice was significantly reduced compared with those treated with a control protein. CD4(+) T cells of lymph nodes and spleen from Foxp3-11R treated mice showed increased levels of Foxp3 expression compared with those of a control protein treated. These results demonstrated that Foxp3-11R can enhance T cell suppressive function and ameliorate experimental arthritis and suggest that cell penetrating recombinant Foxp3 is a potentially useful agent in therapy of arthritis.
Assuntos
Artrite Experimental/terapia , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Proliferação de Células , Meios de Cultura/metabolismo , Feminino , Fatores de Transcrição Forkhead/administração & dosagem , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/uso terapêutico , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Peptídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Baço/metabolismo , Baço/patologia , Zimosan/efeitos adversosRESUMO
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. METHODS: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. RESULTS: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. CONCLUSION: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.
Assuntos
Engenharia Celular/métodos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Fatores de Transcrição Forkhead/administração & dosagem , Terapia Genética/métodos , Receptores de Antígenos de Linfócitos T/administração & dosagem , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Administração Intranasal , Animais , Linhagem Celular , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/genética , Feminino , Fatores de Transcrição Forkhead/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Lentivirus/genética , Lentivirus/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Reguladores/transplanteRESUMO
Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4(+)CD25(-) T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.