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1.
Mol Ther ; 29(1): 86-102, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33010230

RESUMO

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Doença Granulomatosa Crônica/complicações , Doença Granulomatosa Crônica/terapia , Neoplasias Hematológicas/etiologia , Lentivirus/genética , Animais , Modelos Animais de Doenças , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Doença Granulomatosa Crônica/genética , Humanos , Camundongos , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Fatores de Tempo , Resultado do Tratamento
2.
Proc Natl Acad Sci U S A ; 116(30): 15140-15149, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31182588

RESUMO

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in "at-risk" individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.


Assuntos
Colite/imunologia , Diabetes Mellitus Tipo 1/genética , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Ilhotas Pancreáticas/imunologia , Linfócitos T/imunologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Glicemia/imunologia , Glicemia/metabolismo , Colite/induzido quimicamente , Colite/patologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Permeabilidade , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Dodecilsulfato de Sódio/administração & dosagem , Análise de Sobrevida , Linfócitos T/patologia , Transgenes
3.
Int J Mol Sci ; 23(22)2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36430961

RESUMO

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.


Assuntos
Fibrose Cística , Cistos , Furocumarinas , Humanos , Fibrose Cística/genética , NF-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Furocumarinas/farmacologia , Cistos/tratamento farmacológico , Pseudomonas aeruginosa/metabolismo
4.
J Vasc Bras ; 20: e20200122, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-34093684

RESUMO

External iliac artery endofibrosis is a rare pathology that affects high-level endurance athletes, especially cyclists. Classical symptoms include pain, loss of power, and/or cramp in the affected limb while training at maximal effort. The patient's lack of atherosclerotic risk factors makes clinical suspicion of arteriopathy challenging. Moreover, the best management of such patients is still a subject of discussion. We report the case of a 36-year-old professional female endurance cyclist who presented with lower extremity pain during training. Right external iliac artery endofibrosis was diagnosed and the patient underwent surgical treatment. At two-months follow-up, she reported significant improvement in symptoms. This case highlights the importance of diagnosing peripheral vascular disease in young patients and athletes, who do not fit the ordinary profile of patients with atherosclerotic risk factors.


A endofibrose da artéria ilíaca externa é uma doença rara que afeta atletas de resistência (endurance) de nível competitivo, principalmente ciclistas. Os sintomas clássicos incluem dor, perda de força e/ou câimbras no membro afetado ao fazer esforço máximo durante o treino. A ausência de fatores de risco ateroscleróticos nesses pacientes torna a suspeita clínica de arteriopatia desafiadora. Além disso, o melhor manejo ainda é motivo de debate. Relatamos o caso de uma ciclista de resistência profissional, de 36 anos, que apresentou dor nos membros inferiores durante o treino. Foi diagnosticada endofibrose da artéria ilíaca externa, e a paciente foi submetida a tratamento cirúrgico. Após seguimento por dois meses, a paciente relatou melhora significativa dos sintomas. Este caso destaca a importância do diagnóstico de doença vascular periférica em pacientes jovens e atletas, os quais não se encaixam no perfil comum do paciente com fatores de risco ateroscleróticos.

5.
J Vasc Bras ; 20: e20200243, 2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34104134

RESUMO

Isolated dissection of the internal carotid artery (ICA) is rare in young patients and is a cause for strong suspicion of fibromuscular dysplasia (FMD), especially when associated with artery elongation and tortuosity. The natural history of cerebrovascular FMD is unknown and management of symptomatic patients can be challenging. We report the case of a 44-year-old female patient with a history of transient ischemic attack in the absence of cardiovascular risk factors, associated with an isolated left ICA dissection and kinking. Carotid duplex ultrasound confirmed the diagnosis of dissection and demonstrated severe stenosis of the left ICA. The patient underwent surgical repair and histopathological evaluation confirmed the diagnosis of FMD with dissection. An autogenous great saphenous vein bypass was performed and the patient had an uneventful recovery. Cervical carotid artery dissection can be related to underlying arterial pathologies such as FMD, and the presence of ICA tortuosity highlights certain peculiarities for optimal management, which might be surgical.


A dissecção isolada da artéria carótida interna em pacientes jovens é rara, e a displasia fibromuscular deve ser altamente suspeitada principalmente quando estiver associada a alongamento e tortuosidade da artéria. A história natural da displasia fibromuscular cerebrovascular é desconhecida, e o manejo de pacientes sintomáticos pode ser desafiador. Apresentamos o caso de uma paciente de 44 anos com histórico de ataque isquêmico transitório sem fatores de risco cardiovasculares, associado a dissecção e acotovelamento isolados da artéria carótida interna esquerda. O ultrassom duplo das carótidas confirmou o diagnóstico de dissecção e demonstrou estenose grave na artéria carótida interna esquerda. A paciente foi submetida a reparo cirúrgico, e a avaliação histopatológica confirmou o diagnóstico de displasia fibromuscular com dissecção. Foi realizada cirurgia de ressecção do segmento e reconstrução com veia safena magna autógena, e a paciente se recuperou sem complicações. A dissecção da artéria carótida cervical pode estar relacionada a doenças arteriais subjacentes, como a displasia fibromuscular, e a presença da tortuosidade da artéria carótida interna destaca algumas particularidades no manejo ideal, o qual pode ser cirúrgico.

6.
J Card Surg ; 35(9): 2418-2421, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32627263

RESUMO

Mesothelial or monocytic incidental cardiac excrescence (MICE) is a rare, benign lesion composed of a mixture of mesothelial and inflammatory cells, histiocytes and fibrin without a vascular network usually accidentally found during cardiac surgery or after cardiac catherization. We report the case of a 45-year-old man admitted for suspected myxoma on the mitral valve. The lesion was removed via a minimally invasive video-assisted approach, without compromising the valve competency. The histopathological exam led to the diagnosis of MICE. The 2-year follow-up echocardiography confirmed normal mitral function without recurrence. Most of the cases were treated concomitantly to other cardiac procedure, lesions were more frequently founded in the left cardiac chambers or valves, our experience suggest that these lesions should be safely treated alone by surgical removal, to prevent embolic events.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Valva Mitral , Epitélio , Histiócitos , Humanos , Valva Mitral/diagnóstico por imagem , Valva Mitral/cirurgia , Recidiva Local de Neoplasia
7.
Int J Mol Sci ; 21(18)2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32916885

RESUMO

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades, affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge, we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection for up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of lymphocytes, aggregates of vacuolated histiocytes and a few neutrophils, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.


Assuntos
Modelos Animais de Doenças , Pulmão/patologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus , Pneumonia Bacteriana/patologia , Animais , Doença Crônica , Pulmão/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Infecções por Mycobacterium não Tuberculosas/diagnóstico por imagem , Pneumonia Bacteriana/diagnóstico por imagem
8.
J Allergy Clin Immunol ; 142(6): 1909-1921.e9, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29705245

RESUMO

BACKGROUND: Forkhead box P3 (FOXP3) is a key transcription factor in regulatory T (Treg) cell function. FOXP3 gene mutations cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, a fatal autoimmune syndrome. FOXP3 has also been proposed to act in effector T (Teff) cells, but to date, this role has not been confirmed. OBJECTIVE: We sought to evaluate the effect of reduced FOXP3 expression on human Treg and Teff cell development and correlate it with IPEX syndrome immune pathology. METHODS: We developed a model of humanized mice (huMice) in which the human hematopoietic system is stably knocked down or knocked out for the FOXP3 gene (knockdown [KD]/knockout [KO] huMice). RESULTS: Because FOXP3-KD/KO was not 100% effective, residual FOXP3 expression in hematopoietic stem progenitor cells was sufficient to give rise to Treg cells with normal expression of FOXP3. However, numerous defects appeared in the Teff cell compartment. Compared with control mice, FOXP3-KD/KO huMice showed altered thymocyte differentiation, with KD/KO thymocytes displaying significantly reduced T-cell receptor (TCR) signaling strength and increased TCR repertoire diversity. Peripheral KD/KO Teff cells were expanded and showed signs of homeostatic proliferation, such as a significantly contracted TCR repertoire, a severely reduced naive compartment, decreased telomeric repeat-binding factor 2 expression, and a skew toward a TH2 profile, resembling an aged immune system. Consistent with results in FOXP3-KD/KO huMice, analysis of patients with IPEX syndrome provided evidence of defects in the Teff cell compartment at both the thymic and peripheral levels. CONCLUSIONS: These findings support an intrinsic role for human FOXP3 in controlling thymocyte maturation and peripheral expansion of Teff cells and reveal a previously undescribed pathogenic mechanism through an altered Teff cell compartment in patients with IPEX syndrome.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Fatores de Transcrição Forkhead/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T/imunologia , Timo/imunologia , Adolescente , Adulto , Animais , Diferenciação Celular , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/imunologia , Homeostase , Humanos , Doenças do Sistema Imunitário/imunologia , Lactente , Recém-Nascido , Masculino , Camundongos Transgênicos , Adulto Jovem
9.
J Immunol ; 197(7): 2583-8, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27566822

RESUMO

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FcεRI. Tumor protection was also lost after depletion of CD8(+) T cells, highlighting a cross-talk between IgE and T cell-mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology.


Assuntos
Imunoglobulina E/imunologia , Vigilância Imunológica/imunologia , Neoplasias/imunologia , Receptores de IgE/imunologia , Adjuvantes Imunológicos , Animais , Engenharia Genética , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Receptores de IgE/deficiência
10.
Biochim Biophys Acta Gen Subj ; 1861(2): 354-364, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27913191

RESUMO

BACKGROUND: The membrane-bound isoform of the receptor for advanced glycation end products (FL-RAGE) is primarily expressed by alveolar epithelial cells and undergoes shedding by the protease ADAM10, giving rise to soluble cleaved RAGE (cRAGE). RAGE has been associated with the pathogenesis of several acute and chronic lung disorders. Whether the proteolysis of FL-RAGE is altered by a given inflammatory stimulus is unknown. Pseudomonas aeruginosa causes nosocomial infections in hospitalized patients and is the major pathogen associated with chronic lung diseases. METHODS: P. aeruginosa was injected in Rage-/- and wild-type mice and the impact on RAGE expression and shedding, levels of inflammation and bacterial growth was determined. RESULTS: Acute P. aeruginosa lung infection in mice induces a reduction of the active form of ADAM10, which determines an increase of FL-RAGE expression on alveolar cells and a concomitant decrease of pulmonary cRAGE levels. This was associated with massive recruitment of leukocytes and release of pro-inflammatory factors, tissue damage and relocation of cRAGE in the alveolar and bronchial cavities. The administration of sRAGE worsened bacterial burden and neutrophils infiltration. RAGE genetic deficiency reduced the susceptibility to P. aeruginosa infection, mitigating leukocyte recruitment, inflammatory molecules production, and bacterial growth. CONCLUSIONS: These data are the first to suggest that inhibition of FL-RAGE shedding, by affecting the FL-RAGE/cRAGE levels, is a novel mechanism for controlling inflammation to acute P. aeruginosa pneumonia. sRAGE in the alveolar space sustains inflammation in this setting. GENERAL SIGNIFICANCE: RAGE shedding may determine the progression of inflammatory lung diseases.


Assuntos
Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína ADAM10/metabolismo , Animais , Produtos Finais de Glicação Avançada/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo
11.
Mol Ther ; 24(10): 1873-1880, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27456061

RESUMO

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus. Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20-98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus, significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.


Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/complicações , Transplante de Células-Tronco Hematopoéticas/métodos , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Pneumonia Estafilocócica/terapia , Staphylococcus aureus/fisiologia , Animais , Carga Bacteriana , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Doença Granulomatosa Crônica/genética , Células-Tronco Hematopoéticas/virologia , Humanos , Lentivirus/genética , Camundongos , NADPH Oxidase 2 , Pneumonia Estafilocócica/genética , Pneumonia Estafilocócica/microbiologia
13.
Nat Methods ; 10(2): 155-61, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23314173

RESUMO

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.


Assuntos
Carcinoma Hepatocelular/genética , Lentivirus/genética , Neoplasias Hepáticas/genética , Mutagênese Insercional , Oncogenes , Animais , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Vetores Genéticos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/deficiência , Pré-Albumina/genética , Receptor de Interferon alfa e beta/deficiência
14.
J Immunol ; 192(1): 523-32, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24277698

RESUMO

Cancer-initiating cells (CICs) that are responsible for tumor initiation, propagation, and resistance to standard therapies have been isolated from human solid tumors, including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display "tumor-initiating/stemness" properties, including the expression of CIC-associated markers (e.g., CD44, CD24, ALDH-1, EpCAM, Lgr5), multipotency, and tumorigenicity following injection in immunodeficient mice. The immune profile of these cells was assessed by phenotype analysis and by in vitro stimulation of PBMCs with CICs as a source of Ags. CICs, compared with non-CIC counterparts, showed weak immunogenicity. This feature correlated with the expression of high levels of immunomodulatory molecules, such as IL-4, and with CIC-mediated inhibitory activity for anti-tumor T cell responses. CIC-associated IL-4 was found to be responsible for this negative function, which requires cell-to-cell contact with T lymphocytes and which is impaired by blocking IL-4 signaling. In addition, the CRC-associated Ag COA-1 was found to be expressed by CICs and to represent, in an autologous setting, a target molecule for anti-tumor T cells. Our study provides relevant information that may contribute to designing new immunotherapy protocols to target CICs in CRC patients.


Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Vigilância Imunológica/imunologia , Interleucina-4/metabolismo , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Linfócitos T/imunologia , Evasão Tumoral/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Interleucina-4/antagonistas & inibidores , Ativação Linfocitária/imunologia , Esferoides Celulares , Células Tumorais Cultivadas
15.
J Virol ; 88(7): 3623-35, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24403594

RESUMO

UNLABELLED: The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. IMPORTANCE: The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/administração & dosagem , Imunização/métodos , Imunoglobulina A/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores CCR5/imunologia , Receptores de HIV/imunologia , Adjuvantes Imunológicos/administração & dosagem , Estruturas Animais/patologia , Animais , Autoantígenos/imunologia , Linfócitos B/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Portadores de Fármacos , Histocitoquímica , Camundongos , Nodaviridae/genética , Nodaviridae/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia
16.
Hepatology ; 59(6): 2331-43, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24452456

RESUMO

UNLABELLED: Aberrant DNA replication induced by deregulated or excessive proliferative stimuli evokes a "replicative stress response" leading to cell cycle restriction and/or apoptosis. This robust fail-safe mechanism is eventually bypassed by transformed cells, due to ill-defined epistatic interactions. The COP9 signalosome (CSN) is an evolutionarily conserved regulator of cullin ring ligases (CRLs), the largest family of ubiquitin ligases in metazoans. Conditional inactivation of the CSN in several tissues leads to activation of S- or G2-phase checkpoints resulting in irreversible cell cycle arrest and cell death. Herein we ablated COPS5, the CSNs catalytic subunit, in the liver, to investigate its role in cell cycle reentry by differentiated hepatocytes. Lack of COPS5 in regenerating livers causes substantial replicative stress, which triggers a CDKN2A-dependent genetic program leading to cell cycle arrest, polyploidy, and apoptosis. These outcomes are phenocopied by acute overexpression of c-Myc in COPS5 null hepatocytes of adult mice. CONCLUSION: We propose that combined control of proto-oncogene product levels and proteins involved in DNA replication origin licensing may explain the deleterious consequences of CSN inactivation in regenerating livers and provide insight into the pathogenic role of the frequently observed overexpression of the CSN in hepatocellular carcinoma.


Assuntos
Hepatócitos/fisiologia , Regeneração Hepática , Complexos Multiproteicos/fisiologia , Peptídeo Hidrolases/fisiologia , Animais , Complexo do Signalossomo COP9 , Replicação do DNA , Feminino , Genes myc , Genes p16 , Homeostase , Fígado/fisiologia , Fígado/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Poliploidia
17.
Mol Ther ; 22(4): 774-85, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24441399

RESUMO

Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.


Assuntos
Carcinogênese/genética , Terapia Genética , Vetores Genéticos/efeitos adversos , Lentivirus/genética , Vetores Genéticos/uso terapêutico , Humanos , Lentivirus/patogenicidade , Mutagênese Insercional/genética , Regiões Promotoras Genéticas
18.
Blood ; 120(23): 4517-20, 2012 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-23043073

RESUMO

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.


Assuntos
Fator IX/genética , Terapia Genética/métodos , Hemofilia B/terapia , Mutação , Substituição de Aminoácidos , Animais , Coagulação Sanguínea/genética , Coagulação Sanguínea/fisiologia , Cães , Fator IX/fisiologia , Estudos de Viabilidade , Vetores Genéticos/genética , Hemofilia B/genética , Humanos , Lentivirus/genética , Camundongos , Tempo de Tromboplastina Parcial , Resultado do Tratamento
19.
Blood ; 119(6): 1428-39, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22184407

RESUMO

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.


Assuntos
5'-Nucleotidase/imunologia , Adenosina/imunologia , Agamaglobulinemia/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Imunodeficiência Combinada Severa/imunologia , Linfócitos T Reguladores/imunologia , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Adenosina Desaminase/uso terapêutico , Adolescente , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Autoanticorpos/imunologia , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Hipotireoidismo/enzimologia , Hipotireoidismo/genética , Hipotireoidismo/imunologia , Imuno-Histoquímica , Lactente , Masculino , Camundongos , Camundongos Knockout , Polietilenoglicóis/química , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Linfócitos T Reguladores/metabolismo
20.
Invest New Drugs ; 32(6): 1123-33, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25134489

RESUMO

The anticancer activity of a novel pure 1,4-Diaryl-2-azetidinone (1), endowed with a higher solubility than the well known Combretastatin A4, is tested in mice. We previously reported that Compound (1) showed specific antiproliferative activity against duodenal and colon cancer cells, inducing activation of AMP-activated protein kinase and apoptosis. Here we estimate that the maximum tolerated dose in a mouse model is 40 mg/kg; the drug is well tolerated both in single dose and in repeated administration schedules. The drug displays a significant antitumor activity and a tumor growth delay when administered at the MTD both in single and fractionated i.v. administration in a mouse xenograft model of colorectal cancer. Arrest of tumor growth and relapse after drug suspension are parallel to modification in glucose demand as shown by PET studies with [(18)F] FDG. These data strongly support Compound (1) as a promising molecule for in vivo treatment of colorectal cancer.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos , Azetidinas , Neoplasias Colorretais/tratamento farmacológico , Glucose/metabolismo , Guaiacol/análogos & derivados , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Azetidinas/sangue , Azetidinas/farmacocinética , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Guaiacol/sangue , Guaiacol/farmacocinética , Guaiacol/farmacologia , Guaiacol/uso terapêutico , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
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