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1.
Proteomics ; 21(13-14): e2000080, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34081834

RESUMO

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.


Assuntos
Vesículas Extracelulares , Proteômica , Meios de Cultura , Células Epiteliais , Humanos , Proteoma
2.
Invest New Drugs ; 30(6): 2236-51, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22359217

RESUMO

Persistent infections with certain high-risk human papillomavirus (HPV) types such as 16 and 18 can result in the development of cervical cancer. Neither of the two prophylactic vaccines against HPV16 and 18 that are in current use have any therapeutic efficacy for prevalent HPV infections. Ablative therapy is widely used for the treatment of HPV cervical dysplasia however disease recurrence is a widely recognized problem. Thus there is a continuing need for therapeutic approaches for the treatment of HPV infections. The HPV16 E6 viral oncoprotein represses surface expression of the cellular adhesion molecule, E-cadherin. Reduced E-cadherin expression on HPV-infected keratinocytes is associated with lowered numbers of antigen-presenting Langerhans cells in the infected epidermis, potentially reducing immune surveillance for HPV. Four chemicals reported to up-regulate E-cadherin were screened for their ability to counteract E6 repression of surface E-cadherin. 5-Aza-2'-deoxycytidine (AzaDC), a DNA methyltransferase inhibitor, and Indole-3-carbinol (I3C), reported to increase E-cadherin through a p21(Waf1/Cip1)-dependent mechanism, had low cytotoxicity and increased or restored E-cadherin expression and adhesive function in HPV16 E6 expressing HCT116 cells. Doxorubicin, also known to induce p21(Waf1/Cip1), increased E-cadherin in E6 expressing cells but had some associated cytotoxicity. Tamoxifen, which can restore adhesive function of surface E-cadherin, was ineffective in counteracting E6 repression of E-cadherin. AzaDC and I3C both show potential to restore antigen-presenting cells to HPV infected skin by antagonizing E6 repression of E-cadherin, thereby counteracting an important immune evasion mechanism of HPV16 and reinstating immune function at the infected site.


Assuntos
Azacitidina/análogos & derivados , Caderinas/metabolismo , Doxorrubicina/farmacologia , Indóis/farmacologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Tamoxifeno/farmacologia , Antivirais , Azacitidina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Células HCT116 , Humanos
3.
J Immunol Methods ; 302(1-2): 116-24, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15992814

RESUMO

Epithelial (E)-cadherin is a transmembrane protein that mediates calcium-dependent cell adhesion. E-cadherin has significant roles in tissue development, adhesion between keratinocytes and retention of Langerhans cells in the epidermis, and its loss on tumour cells is frequently associated with metastasis. Here we describe a simple, flow cytometric adhesion assay to measure the effects of potential regulators of cell surface E-cadherin expression on E-cadherin-mediated adhesion between cells. In this assay, cells that have been transiently transfected to express the protein of interest are enzymatically treated to remove cell surface E-cadherin. Cells are then incubated in low attachment plates, during which time the E-cadherin is re-expressed and E-cadherin-mediated aggregation occurs. The effect of the protein of interest on the percentage of E-cadherin-mediated aggregates that form during incubation is measured flow cytometrically, following staining with an E-cadherin specific antibody. A major advantage of this assay is that a potential regulatory protein of interest can be tested in a transient expression system by co-expression with green fluorescent protein and analysis of adhesion conducted on green fluorescent cells only. We have applied this assay to measure the regulatory effects of E6 protein from human papillomavirus type 16 on E-cadherin-mediated adhesion but this assay potentially has broad applicability for testing the effects of other proteins on E-cadherin-mediated adhesion in an accurate and highly reproducible manner.


Assuntos
Caderinas/fisiologia , Citometria de Fluxo/métodos , Adesão Celular/genética , Agregação Celular/genética , Linhagem Celular Transformada , Genes Reporter , Humanos , Queratinócitos/fisiologia , Proteínas Oncogênicas Virais/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/fisiologia , Transfecção
4.
J Leukoc Biol ; 97(1): 103-10, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25416814

RESUMO

Macrophage adherence to the inner corneal surface and formation of MGCs in the stroma are common signs of chronic inflammation following corneal infection. To determine whether macrophage adherence (known clinically as KPs) and giant cell formation were specific to innate immune activation via particular TLR ligands, macrophage activation was examined in a murine model of TLR-mediated corneal inflammation. The corneal epithelium was debrided and highly purified TLR ligands were topically applied once to the cornea of TLR7(-/-), TLR9(-/-), Cx3cr1(gfp/+), CD11c(eYFP), and IL-4(-/-) mice. At 1 week post-treatment macrophage activation and phenotype was evaluated in the cornea. Treatment with TLR2, TLR3, TLR4, and TLR5 ligands caused an increase in the number of activated stromal macrophages in the central cornea at 1 week post-treatment. However, treatment with TLR9 ligand CpG-ODN and the TLR7/8 ligand R848/Resiquimod led to an accumulation of macrophages on the corneal endothelium and formation of multinucleated giant macrophages in the corneal stroma. We suggest that giant cell formation, which is a characteristic feature of granuloma formation in many tissues, may be a unique feature of TLR9- and TLR7/8-mediated macrophage activation.


Assuntos
Córnea/imunologia , Ceratite/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Células Gigantes/imunologia , Células Gigantes/metabolismo , Ceratite/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo
5.
J Invest Dermatol ; 130(2): 472-80, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19759549

RESUMO

Human papillomaviruses (HPVs), which are contained in the alpha, beta, gamma, mu, and nu genera, differ in their oncogenic potential and their tropism for cutaneous or mucosal epidermis. Langerhans cells (LC), the only epidermal professional antigen-presenting cells, are readily detected in normal mucosal and cutaneous epithelium. The aim of this study is to determine whether LC loss, which has been reported for HPV16, occurs in other HPV genera and establish its significance in viral pathology. We found that, as for HPV16, LCs were reduced in lesions infected with high-risk mucosal (alpha7 and alpha9 species) and low-risk cutaneous (gamma and mu) types. Lesions infected with alpha10 low-risk genital types had reduced LC but contained epidermal LC patches, coincident with dermis-localized regulatory T cells (T-regs). In contrast to other genera, LCs were common in the epidermis, and T-regs occupied the dermis of the potentially high-risk cutaneous beta-HPV type infected lesions. Therefore, LC loss in the infected lesions occurred irrespective of tropism or oncogenic potential of the HPV type. LC depletion in the HPV-infected epidermis may create an environment that is permissive for viral persistence and in HPV lesions in which LCs are found, the presence of typically immunosuppressive T-regs may compensate for their continued presence.


Assuntos
Células Epidérmicas , Células de Langerhans/citologia , Papillomaviridae/metabolismo , Infecções por Papillomavirus/virologia , Células Apresentadoras de Antígenos/citologia , Antígenos CD1/biossíntese , Biópsia , Condiloma Acuminado/virologia , Epiderme/virologia , Humanos , Sistema Imunitário , Células de Langerhans/virologia , Mucosa/virologia , Risco , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/virologia
6.
J Virol ; 77(15): 8378-85, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12857907

RESUMO

Human papillomavirus type 16 (HPV16) is an oncogenic virus that causes persistent infections in cervical epithelium. The chronic nature of HPV16 infections suggests that this virus actively evades the host immune response. Intraepithelial Langerhans cells (LC) are antigen-presenting cells that are critical in T-cell priming in response to viral infections of the skin. Here we show that HPV16 infection is directly associated with a reduction in the numbers of LC in infected epidermis. Adhesion between keratinocytes (KC) and LC, mediated by E-cadherin, is important in the retention of LC in the skin. Cell surface E-cadherin is reduced on HPV16-infected basal KC, and this is directly associated with the reduction in numbers of LC in infected epidermis. Expression of a single viral early protein, HPV16 E6, in KC reduces levels of cell surface E-cadherin thereby interfering with E-cadherin-mediated adhesion. Through this pathway, E6 expression in HPV16-infected KC may limit presentation of viral antigens by LC to the immune system, thus preventing the initiation of a cell-mediated immune response and promoting survival of the virus.


Assuntos
Caderinas/metabolismo , Queratinócitos/virologia , Células de Langerhans/patologia , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/virologia , Proteínas Repressoras , Pele/virologia , Adesão Celular , Linhagem Celular , Colo do Útero/virologia , Regulação para Baixo , Feminino , Humanos , Queratinócitos/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Doenças do Colo do Útero/metabolismo , Doenças do Colo do Útero/patologia , Doenças do Colo do Útero/virologia
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