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1.
JCI Insight ; 4(11)2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31167965

RESUMO

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.

2.
Pediatr Dermatol ; 2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30515866

RESUMO

BACKGROUND/OBJECTIVES: Epidermolysis bullosa is a group of diseases caused by mutations in skin structural proteins. Availability of genetic sequencing makes identification of causative mutations easier, and genotype-phenotype description and correlation are important. We describe six patients with a keratin 5 mutation resulting in a glutamic acid to lysine substitution at position 477 (p.Glu477Lys) who have a distinctive, severe and sometimes fatal phenotype. We also perform in silico modeling to show protein structural changes resulting in instability. METHODS: In this case series, we collected clinical data from six patients with this mutation identified from their national or local epidermolysis bullosa databases. We performed in silico modeling of the keratin 5-keratin 14 coil 2B complex using CCBuilder and rendered with Pymol (Schrodinger, LLC, New York, NY). RESULTS: Features include aplasia cutis congenita, generalized blistering, palmoplantar keratoderma, onychodystrophy, airway and developmental abnormalities, and a distinctive reticulated skin pattern. Our in silico model of the keratin 5 p.Glu477Lys mutation predicts conformational change and modification of the surface charge of the keratin heterodimer, severely impairing filament stability. CONCLUSIONS: Early recognition of the features of this genotype will improve care. In silico analysis of mutated keratin structures provides useful insights into structural instability.

3.
Methods Mol Biol ; 1828: E1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30488375

RESUMO

The original version of this book was published with the following errors: "2'MOE" have been corrected into "2'MOEPS" in figure.6 - Chapter 35, multiple typo errors in page numbers: 532, 533, 534, 537, 542, 548 and 549. These errors has been updated.

4.
Methods Mol Biol ; 1828: 531-552, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171566

RESUMO

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe skin genetic disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon skipping strategy to reframe transcripts.

5.
Sci Transl Med ; 10(455)2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135250

RESUMO

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

6.
Sci Immunol ; 3(24)2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907691

RESUMO

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

8.
J Invest Dermatol ; 137(5): e123-e129, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28411843

RESUMO

Stunning technological advances in genomics have led to spectacular breakthroughs in the understanding of the underlying defects, biological pathways and therapeutic targets of skin diseases leading to new therapeutic interventions. Next-generation sequencing has revolutionized the identification of disease-causing genes and has a profound impact in deciphering gene and protein signatures in rare and frequent skin diseases. Gene addition strategies have shown efficacy in junctional EB and in recessive dystrophic EB (RDEB). TALENs and Cripsr/Cas9 have emerged as highly efficient new tools to edit genomic sequences to creat new models and to correct or disrupt mutated genes to treat human diseases. Therapeutic approaches have not been limited to DNA modification and strategies at the mRNA, protein and cellular levels have also emerged, some of which have already proven clinical efficacy in RDEB. Improved understanding of the pathogenesis of skin disorders has led to the development of specific drugs or repurposing of existing medicines as in basal cell nevus syndrome, alopecia areata, melanoma and EB simplex. These discoveries pave the way for improved targeted personalized medicine for rare and frequent diseases. It is likely that a growing number of orphan skin diseases will benefit from combinatory new therapies in a near future.


Assuntos
Edição de Genes/métodos , Genômica/métodos , Dermatopatias/genética , Animais , Dermatologia/métodos , Desenho de Drogas , Reposicionamento de Medicamentos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Medicina de Precisão/métodos , Doenças Raras/genética , Doenças Raras/fisiopatologia , Doenças Raras/terapia , Dermatopatias/fisiopatologia , Dermatopatias/terapia
9.
J Invest Dermatol ; 136(12): 2387-2395, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27498345

RESUMO

Dystrophic epidermolysis bullosa is a group of orphan genetic skin diseases dominantly or recessively inherited, caused by mutations in COL7A1 encoding type VII collagen, which forms anchoring fibrils. Individuals with recessive dystrophic epidermolysis bullosa develop severe skin and mucosal blistering after mild trauma. The exon skipping strategy consists of modulating splicing of a pre-mRNA to induce skipping of a mutated exon. We have targeted COL7A1 exons 73 and 80, which carry recurrent mutations and whose excision preserves the open reading frame. We first showed the dispensability of these exons for type VII collagen function in vivo. We then showed that transfection of primary recessive dystrophic epidermolysis bullosa keratinocytes and fibroblasts carrying null mutations in exon 73 and/or 80, with 2'-O-methyl antisense oligoribonucleotides, led to efficient ex vivo skipping of these exons (50-95%) and resulted in a significant level (up to 36%) of type VII collagen re-expression. Finally, one or two subcutaneous injections of antisense oligoribonucleotides at doses ranging from 400 µg up to 1 mg restored type VII collagen expression and anchoring fibril formation in vivo in a xenograft model of recessive dystrophic epidermolysis bullosa skin equivalent. This work provides a proof of principle for the treatment of patients with recessive dystrophic epidermolysis bullosa by exon skipping using subcutaneous administration of antisense oligoribonucleotides.


Assuntos
Colágeno Tipo VII/genética , Colágeno/genética , Epidermólise Bolhosa Distrófica/genética , Predisposição Genética para Doença , Animais , Western Blotting , Células Cultivadas , Complemento C7 , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/fisiopatologia , Éxons/genética , Regulação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Nus , Processamento de RNA/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transfecção
10.
J Am Acad Dermatol ; 74(6): 1166-72, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26947449

RESUMO

BACKGROUND: Serologic diagnosis of epidermolysis bullosa acquisita (EBA) relies on the detection of circulating autoantibodies to type VII collagen (C7). OBJECTIVE: We sought to compare the diagnostic performances of a commercialized enzyme-linked immunosorbent assay (ELISA) using C7 noncollagenous (NC) domains (C7-NC1/NC2 ELISA) and indirect immunofluorescence (IIF) biochip test on NC1-C7-expressing transfected cells (IIFT), with a full-length-C7 ELISA developed in our laboratory. METHODS: C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were run on 77 nonselected consecutive EBA sera. RESULTS: C7-NC1/NC2 ELISA, IIFT, and full-length-C7 ELISA were positive, respectively, for: 30%, 27%, and 65% of the 77 sera; 43%, 32%, and 80% of 44 sera labeling the salt-split-skin (SSS) floor (F) by IIF (SSS/F(+)); 9%, 22%, and 47% of 32 SSS/F(-) sera; 28%, 28%, and 58% of classic EBA; 41%, 41%, and 82% of inflammatory EBA; and 18%, 0%, and 55% of mucous-membrane-predominant EBA. Significant differences for all sera were found between: the 2 ELISAs for the 77 sera, SSS/F(+) and SSS/F(-) sera, and IIFT versus full-length-C7 ELISA. LIMITATIONS: The retrospective design was a limitation. CONCLUSION: C7-NC1/NC2 ELISA and IIFT sensitivities for serologic diagnoses of EBA were low. Full-length-C7 ELISA was significantly more sensitive and could serve as a reference test.


Assuntos
Autoanticorpos/sangue , Colágeno Tipo VII/imunologia , Epidermólise Bolhosa Adquirida/sangue , Epidermólise Bolhosa Adquirida/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Serial de Proteínas , Curva ROC , Estudos Retrospectivos , Adulto Jovem
11.
J Invest Dermatol ; 136(7): 1346-1354, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26994967

RESUMO

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack type VII collagen and therefore have severely impaired dermal-epidermal stability causing recurrent skin and mucosal blistering. There is currently no specific approved treatment for RDEB. We present preclinical data showing that intradermal injections of genetically corrected patient-derived RDEB fibroblasts using a Good Manufacturing Practices grade self-inactivating COL7A1 retroviral vector reverse the disease phenotype in a xenograft model in nude mice. We obtained 50% transduction efficiency in primary human RDEB fibroblasts with an average low copy number (range = 1-2) of integrated provirus. Transduced fibroblasts showed strong type VII collagen re-expression, improved adhesion properties, normal proliferative capabilities, and viability in vitro. We show that a single intradermal injection of 3 × 10(6) genetically corrected RDEB fibroblasts beneath RDEB skin equivalents grafted onto mice allows type VII collagen deposition, anchoring fibril formation at the dermal-epidermal junction, and improved dermal-epidermal adherence 2 months after treatment, supporting functional correction in vivo. Gene-corrected fibroblasts previously showed no tumorigenicity. These data show the efficacy and safety of gene-corrected fibroblast therapy using a self-inactivating vector that has now been good manufacturing grade-certified and pave the way for clinical translation to treat nonhealing wounds in RDEB patients.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Fibroblastos/metabolismo , Terapia Genética , Animais , Adesão Celular , Proliferação de Células , Colágeno Tipo VII/metabolismo , Fibroblastos/citologia , Genes Recessivos , Vetores Genéticos , Células HEK293 , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Proteínas Recombinantes/genética , Retroviridae , Pele/metabolismo , Temperatura Ambiente
12.
Hum Gene Ther Clin Dev ; 25(4): 218-28, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25381930

RESUMO

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.


Assuntos
Colágeno Tipo VII/genética , DNA Recombinante/genética , Gammaretrovirus/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Colágeno Tipo VII/metabolismo , DNA Recombinante/isolamento & purificação , Gammaretrovirus/metabolismo , Marcação de Genes/métodos , Vetores Genéticos/isolamento & purificação , Células HEK293 , Humanos
13.
Methods Mol Biol ; 867: 221-38, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22454065

RESUMO

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Éxons , Mutação , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Linhagem Celular , Clonagem Molecular/métodos , DNA Complementar/genética , Epidermólise Bolhosa Distrófica/terapia , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Oligonucleotídeos Antissenso/genética , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/isolamento & purificação , Análise de Sequência/métodos , Transfecção
16.
J Invest Dermatol ; 131(4): 848-56, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21124339

RESUMO

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.


Assuntos
Epidermólise Bolhosa Distrófica , Genes Recessivos , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Epigênese Genética/fisiologia , Fibroblastos/citologia , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Medicina de Precisão
17.
Dermatol Clin ; 28(2): 361-6, xii, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20447504

RESUMO

Among the severe genetic disorders of the skin that are suitable for gene and cell therapy, most efforts have been made in the treatment of blistering diseases including dystrophic epidermolysis bullosa. This condition can be recessively or dominantly inherited, depending on the nature and position of the mutation or mutations in the gene encoding type VII collagen. At present, there is no specific treatment for recessive dystrophic epidermolysis bullosa, and gene and cell therapy approaches hold great promise. This article discusses the different gene therapy approaches that have been used for the treatment of this disease and the new perspectives that they open.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Terapia Genética/métodos , Terapia Genética/tendências , Humanos
18.
Mol Ther ; 18(8): 1509-18, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20485266

RESUMO

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in COL7A1 encoding type VII collagen which forms key structures (anchoring fibrils) for dermal-epidermal adherence. Patients suffer since birth from skin blistering, and develop severe local and systemic complications resulting in poor prognosis. We lack a specific treatment for RDEB, but ex vivo gene transfer to epidermal stem cells shows a therapeutic potential. To minimize the risk of oncogenic events, we have developed new minimal self-inactivating (SIN) retroviral vectors in which the COL7A1 complementary DNA (cDNA) is under the control of the human elongation factor 1alpha (EF1alpha) or COL7A1 promoters. We show efficient ex vivo genetic correction of primary RDEB keratinocytes and fibroblasts without antibiotic selection, and use either of these genetically corrected cells to generate human skin equivalents (SEs) which were grafted onto immunodeficient mice. We achieved long-term expression of recombinant type VII collagen with restored dermal-epidermal adherence and anchoring fibril formation, demonstrating in vivo functional correction. In few cases, rearranged proviruses were detected, which were probably generated during the retrotranscription process. Despite this observation which should be taken under consideration for clinical application, this preclinical study paves the way for a therapy based on grafting the most severely affected skin areas of patients with fully autologous SEs genetically corrected using a SIN COL7A1 retroviral vector.


Assuntos
Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/terapia , Vetores Genéticos/genética , Retroviridae/genética , Animais , Southern Blotting , Western Blotting , Células Cultivadas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/metabolismo , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Camundongos , Camundongos SCID , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética
19.
J Clin Immunol ; 30(4): 607-19, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20405178

RESUMO

INTRODUCTION: Bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells share immunosuppressive capacities, suggesting that the latter could be a general property of stromal cells. METHODS: To check this hypothesis, we compared human BM-MSC and fibroblasts for their in vitro multi-potentiality, expandability and their immunomodulatory properties under normalized optimized culture conditions. RESULTS: We report that, unlike BM-MSCs, fibroblasts cannot differentiate in vitro into adipocytes and osteoblasts and differ from BM-MSCs by the expression of membrane CD106, CD10 and CD26 and by the expression of collagen VII mRNA. Like BM-MSCs, fibroblasts are unable to provoke in vitro allogeneic reactions, but strongly suppress lymphocyte proliferation induced by allogeneic mixed lymphocyte culture (MLC) or mitogens. We show that fibroblasts' immunosuppressive capacity is independent from prostaglandin E2, IL-10 and the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase and is not abrogated after the depletion of CD8+ T lymphocytes, NK cells and monocytes. CONCLUSION: Finally, fibroblasts and BM-MSCs act at an early stage through blockage of lymphocyte activation, as demonstrated by down-regulation of GZMB (granzyme B) and IL2RA (CD25) expression.


Assuntos
Fibroblastos/imunologia , Células-Tronco Mesenquimais/imunologia , Tecido Adiposo/citologia , Células da Medula Óssea , Células Cultivadas , Regulação para Baixo/genética , Fibroblastos/citologia , Granzimas/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Ativação Linfocitária , Células-Tronco Mesenquimais/citologia , Células Estromais/imunologia
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