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1.
Nat Immunol ; 16(3): 318-325, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25621826

RESUMO

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.


Assuntos
Fatores de Transcrição Maf/genética , RNA Longo não Codificante/genética , Linfócitos T/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Fatores de Transcrição Maf/imunologia , RNA Longo não Codificante/imunologia , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Transcriptoma/genética , Transcriptoma/imunologia
2.
Immunity ; 45(5): 1135-1147, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851914

RESUMO

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Separação Celular , Neoplasias Colorretais/mortalidade , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Transcriptoma
3.
J Cell Mol Med ; 24(17): 9945-9957, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32666618

RESUMO

The current standard biomarker for myocardial infarction (MI) is high-sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched-controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface-epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non-inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI.


Assuntos
Angina Estável/sangue , Biomarcadores/sangue , Epitopos/sangue , Vesículas Extracelulares/genética , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/metabolismo , Síndrome Coronariana Aguda/patologia , Idoso , Angina Estável/genética , Angina Estável/patologia , Antígenos CD40/sangue , Estudos de Coortes , Mapeamento de Epitopos , Epitopos/genética , Feminino , Humanos , Integrina alfa2/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Intervenção Coronária Percutânea , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Infarto do Miocárdio com Supradesnível do Segmento ST/genética , Infarto do Miocárdio com Supradesnível do Segmento ST/patologia
5.
Blood ; 130(5): 606-618, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28637663

RESUMO

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.


Assuntos
Transferência Adotiva , Edição de Genes/métodos , Memória Imunológica , Mieloma Múltiplo , Proteínas de Neoplasias , Fragmentos de Peptídeos , Receptores de Antígenos de Linfócitos T , Linfócitos T , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Transferência de Genes , Doença Enxerto-Hospedeiro , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Blood ; 121(4): 573-84, 2013 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-23160470

RESUMO

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.


Assuntos
Memória Imunológica , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-15/genética , Interleucina-7/genética , Selectina L/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/transplante , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/transplante , Receptor fas/metabolismo
7.
Nat Methods ; 8(10): 861-9, 2011 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-21857672

RESUMO

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.


Assuntos
Técnicas de Transferência de Genes , Mutagênese Insercional/genética , Mutagênese Sítio-Dirigida , Dependovirus/genética , Humanos , Receptores CCR5/genética , Integração Viral/genética
8.
J Colloid Interface Sci ; 667: 338-349, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38640653

RESUMO

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.


Assuntos
Vesículas Extracelulares , Peptídeos , Humanos , Vesículas Extracelulares/química , Peptídeos/química , Peptídeos/isolamento & purificação , Membranas Artificiais , Tamanho da Partícula , Propriedades de Superfície
9.
Nat Med ; 12(12): 1397-402, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17115047

RESUMO

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.


Assuntos
Células Epidérmicas , Epidermólise Bolhosa Juncional/terapia , Terapia Genética/métodos , Transplante de Células-Tronco , Células 3T3 , Adulto , Animais , Moléculas de Adesão Celular/genética , Células Cultivadas , Estudos de Viabilidade , Vetores Genéticos , Humanos , Masculino , Camundongos , Retroviridae , Engenharia Tecidual/métodos , Calinina
10.
Methods Mol Biol ; 2668: 69-98, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37140791

RESUMO

The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~1013 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas.


Assuntos
Vesículas Extracelulares , Meios de Cultivo Condicionados/análise , Vesículas Extracelulares/química , Filtração , Ultracentrifugação
11.
Blood ; 113(5): 1006-15, 2009 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18978209

RESUMO

Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.


Assuntos
Genes Transgênicos Suicidas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Memória Imunológica , Interleucina-15/farmacologia , Interleucina-7/farmacologia , Transplante de Células-Tronco , Linfócitos T/imunologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Genes Transgênicos Suicidas/genética , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/terapia , Humanos , Memória Imunológica/genética , Interleucina-15/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-7/imunologia , Isoantígenos/genética , Isoantígenos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia
12.
Methods Mol Biol ; 2286: 131-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33381854

RESUMO

Cardiac explant-derived cells (cEDC), also referred as cardiac progenitors cells (CPC) (Barile et al., Cardiovasc Res 103(4):530-541, 2014; Barile et al., Cardiovasc Res 114(7):992-1005, 2018), represent promising candidates for the development of cell-based therapies, a novel and interesting treatment for cardioprotective strategy in heart failure (Kreke et al., Expert Rev Cardiovasc Ther 10(9):1185-1194, 2012). CPC have been tested in a preclinical setting for direct cell transplantation and tissue engineering or as a source for production of extracellular vesicles (EV) (Oh et al., J Cardiol 68(5):361-367, 2016; Barile et al., Eur Heart J 38(18):1372-1379, 2017; Rosen et al., J Am Coll Cardiol 64(9):922-937, 2014). CPC cultured as cardiospheres derived cells went through favorable Phase 1 and 2 studies demonstrating safety and possible efficacy (Makkar et al., Lancet 379(9819):895-904, 2012; Ishigami et al., Circ Res 120(7):1162-1173, 2017; Ishigami et al., Circ Res 116 (4):653-664, 2015; Tarui et al., J Thorac Cardiovasc Surg 150(5):1198-1207, 1208 e1191-1192, 2015). In this context and in view of clinical applications, cells have to be prepared and released according to Good Manufacturing Practices (GMP) (EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part I-basic requirements for medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ; EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part IV-guidelines on good manufacturing practices specific to advanced therapy medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ). This chapter describes GMP-grade methods for production and testing of a CPC Master Cell Bank (MCB), consisting of frozen aliquots of cells that may be used either as a therapeutic product or as source for the manufacturing of Exo for clinical trials.The MCB production method has been designed to isolate and expand CPC from human cardiac tissue in xeno-free conditions (Andriolo et al., Front Physiol 9:1169, 2018). The quality control (QC) methods have been implemented to assess the safety (sterility, endotoxin, mycoplasma, cell senescence, tumorigenicity) and identity/potency/purity (cell count and viability, RT-PCR, immunophenotype) of the cells (Andriolo et al., Front Physiol 9:1169, 2018).


Assuntos
Tecnologia Biomédica/normas , Mioblastos/citologia , Miócitos Cardíacos/citologia , Cultura Primária de Células/métodos , Bancos de Espécimes Biológicos/normas , Tecnologia Biomédica/métodos , Células Cultivadas , Humanos , Guias de Prática Clínica como Assunto , Cultura Primária de Células/normas , Preservação de Tecido/normas
13.
Front Physiol ; 9: 1169, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30197601

RESUMO

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro, and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>1013 particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro, showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.

14.
Chest ; 150(2): e29-32, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27502989

RESUMO

A young woman received a diagnosis of abdominal, sporadic lymphangioleiomyomatosis (LAM) and multiple abdominal lymphangioleiomyomas and was referred for recurrent chylous ascites responding only to a fat-free diet. On admission, pulmonary function test (PFT) results showed a moderate reduction in the transfer factor for carbon monoxide with normal exercise performance. The serum vascular endothelial growth factor D (VEGF-D) level was 2,209 pg/mL. DNA sequences, amplified at loci kg8, D16S3395, D16S3024, D16S521, and D16S291 on chromosome 16p13.3, showed a loss of heterozygosity (LOH) only for kg8. Fat-free total parenteral nutrition in association with sirolimus (2 mg po daily) was initiated. Serum sirolimus levels were maintained at concentrations between 5 and 15 ng/mL. After 1 month, reintroduction of a low-fat oral feeding was achieved without recurrence of ascites. PFT results were stable. Interestingly, clinical improvement was associated with a reduction in the VEGF-D serum level (1,558 pg/mL). LOH at the kg8 biomarker in blood LAM cells was no longer detected.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Ascite Quilosa/dietoterapia , Neoplasias Pulmonares/tratamento farmacológico , Linfangioleiomiomatose/tratamento farmacológico , Neoplasias Retroperitoneais/tratamento farmacológico , Sirolimo/uso terapêutico , Adulto , Ascite Quilosa/etiologia , Dieta com Restrição de Gorduras , Feminino , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/fisiopatologia , Linfangioleiomiomatose/sangue , Linfangioleiomiomatose/complicações , Linfangioleiomiomatose/diagnóstico por imagem , Imageamento por Ressonância Magnética , Células Neoplásicas Circulantes , Nutrição Parenteral Total , Capacidade de Difusão Pulmonar , Testes de Função Respiratória , Neoplasias Retroperitoneais/sangue , Neoplasias Retroperitoneais/complicações , Neoplasias Retroperitoneais/diagnóstico por imagem , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Fator D de Crescimento do Endotélio Vascular/sangue
15.
Vet Immunol Immunopathol ; 107(1-2): 17-26, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15982476

RESUMO

alpha1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites.


Assuntos
Síndrome de Imunodeficiência Adquirida Felina/metabolismo , Vírus da Imunodeficiência Felina , Vírus da Leucemia Felina , Leucemia Felina/metabolismo , Orosomucoide/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Clonagem Molecular , DNA Complementar/genética , Síndrome de Imunodeficiência Adquirida Felina/genética , Glicosilação , Lectinas/metabolismo , Leucemia Felina/genética , Dados de Sequência Molecular , Orosomucoide/genética , Orosomucoide/metabolismo , Polissacarídeos/química , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos
16.
Front Pharmacol ; 6: 76, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25954199

RESUMO

Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005-2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.

17.
Hum Gene Ther ; 24(2): 220-37, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23311414

RESUMO

Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF(+), IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-α(+), MCP-1(+), HLA-DR(+), CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD(+) AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2rγc(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.


Assuntos
Transferência Adotiva/métodos , Células Dendríticas/imunologia , Genes do Tumor de Wilms , Lentivirus/metabolismo , Leucemia Mieloide Aguda/terapia , Animais , Antineoplásicos/imunologia , Linfócitos T CD8-Positivos , Diferenciação Celular , Sobrevivência Celular , Regulação da Expressão Gênica/imunologia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Lentivirus/genética , Leucemia Mieloide Aguda/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Camundongos , Monócitos , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Risco
18.
Nat Med ; 18(5): 807-815, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22466705

RESUMO

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR ß- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Lentivirus/genética , Leucemia/imunologia , Receptores de Antígenos de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Dedos de Zinco , Sequência de Bases , Técnicas de Transferência de Genes , Humanos , Células Jurkat , Dados de Sequência Molecular , Linfócitos T/imunologia , Proteínas WT1/genética
19.
Hum Gene Ther ; 22(7): 829-41, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21091264

RESUMO

Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (ΔLNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.


Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide Aguda/terapia , Transfusão de Leucócitos , Adulto , Quimerismo , Feminino , Proteínas de Fusão bcr-abl/genética , Vetores Genéticos , Doença Enxerto-Hospedeiro/mortalidade , Humanos , Terapia de Imunossupressão , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Indução de Remissão , Retroviridae/genética , Timidina Quinase/genética , Doadores de Tecidos , Transdução Genética , Transgenes
20.
Hum Gene Ther ; 21(3): 241-50, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20121594

RESUMO

Allogeneic hematopoietic stem cell transplantation (allo-SCT) from an HLA-matched related or unrelated donor is a curative option for patients with high-risk hematological diseases. In the absence of a matched donor, patients have been offered investigational transplantation strategies such as umbilical cord blood SCT or family haploidentical SCT. Besides the activity of the conditioning regimen, most of the antileukemic potential of allo-SCT relies on alloreactivity, promoted by donor lymphocytes reacting against patient-specific antigens, such as minor and major histocompatibility antigens, ultimately translating into cancer immunotherapy. Unfortunately, alloreactivity is also responsible for the most serious and frequent complication of allo-SCT: graft-versus-host-disease (GvHD). The risk of GvHD increases with the level of HLA disparity between host and donor, and leads to impaired quality of life and reduced survival expectancy, particularly among patients receiving transplants from HLA-mismatched donors. Gene transfer technologies are promising tools to manipulate donor T cell immunity to enforce the graft-versus-tumor effect, to promote functional immune reconstitution (graft vs. infection), and to prevent or control GvHD. To this purpose, several cell and gene transfer approaches have been investigated at the preclinical level, and are being implemented in clinical trials. Suicide gene therapy is to date the most extensive clinical application of T cell-based gene therapy. In several phase I-II clinical studies conducted worldwide this approach proved highly feasible, safe, and effective in promoting a dynamic and patient-specific modulation of alloreactivity. This review focuses on this approach.


Assuntos
Genes Transgênicos Suicidas , Terapia Genética , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante Homólogo , Resultado do Tratamento
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