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1.
Nat Commun ; 10(1): 1194, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886143

RESUMO

Chronic infections of the fallopian tubes with Chlamydia trachomatis (Ctr) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation-demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer.


Assuntos
Infecções por Chlamydia/genética , Chlamydia trachomatis/imunologia , Ilhas de CpG/genética , Metilação de DNA/imunologia , Interações entre Hospedeiro e Microrganismos/genética , Células-Tronco/metabolismo , Fatores Etários , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Doença Crônica , Ilhas de CpG/imunologia , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/microbiologia , Epigênese Genética/genética , Epigênese Genética/imunologia , Epitélio/imunologia , Epitélio/metabolismo , Epitélio/microbiologia , Tubas Uterinas/imunologia , Tubas Uterinas/metabolismo , Tubas Uterinas/microbiologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Microscopia Intravital , Microscopia Confocal , Organoides/imunologia , Organoides/metabolismo , Organoides/microbiologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/microbiologia , Sorogrupo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Análise de Célula Única , Células-Tronco/imunologia , Células-Tronco/microbiologia , Técnicas de Cultura de Tecidos
2.
Nat Rev Genet ; 20(7): 377-388, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30737492

RESUMO

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human-rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.


Assuntos
Doenças Transmissíveis/terapia , Doenças Genéticas Inatas/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Neoplasias/terapia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Quimera/genética , Quimera/imunologia , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/patologia , Descoberta de Drogas/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Terapia Genética/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/transplante , Modelos Animais , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/imunologia , Transplante de Tecidos/métodos , Transplante Heterólogo
3.
Cell ; 175(7): 1972-1988.e16, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30550791

RESUMO

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.


Assuntos
Modelos Imunológicos , Neoplasias Experimentais/imunologia , Organoides/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Animais , Antígeno B7-H1/imunologia , Técnicas de Cocultura , Feminino , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Organoides/patologia
4.
Virulence ; 9(1): 231-247, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29099326

RESUMO

Pathogenesis of Staphylococcus aureus is increasingly recognized to be driven by powerful toxins. Staphylococcus aureus employs up to six pore-forming toxins to subvert the human host defense and to promote bacterial invasion: alpha-hemolysin that disrupts epithelial and endothelial barriers and five leukocidins that lyse phagocytes involved in bacterial clearance. Previously, we described two human monoclonal antibodies (mAbs), ASN-1 that neutralizes alpha-hemolysin and four leukocidins (LukSF-PV, LukED, HlgAB, HlgCB), and ASN-2 that inactivates the 5th leukocidin, LukGH. In this study we tested the individual and combined effects of ASN-1 and ASN-2 in multiple in vitro models employing relevant human target cells. We found that diverse S. aureus isolates with different genetic backgrounds (based on MLST- and spa-typing) and antibiotic sensitivity (both MRSA and MSSA) displayed greatly different cytotoxin expression patterns influenced by the type of growth medium used. Both mAbs were required to fully prevent the lysis of human neutrophils exposed to the mixture of recombinant cytotoxins or native toxins present in the culture supernatants of S. aureus isolates. Flow cytometry confirmed the protective effects of ASN-1 + ASN-2 (known as ASN100) on granulocytes, monocytes, NK-cells and T-lymphocytes. ASN-1 alone preserved the integrity of a 3D-primary culture of human tracheal/bronchial mucociliary epithelial tissue infected with S. aureus. We conclude that simultaneous inhibition of alpha-hemolysin and five leukocidins by ASN100 blocks cytolytic activity of S. aureus towards human target cells in vitro.


Assuntos
Anticorpos Monoclonais/imunologia , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Anticorpos Monoclonais/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Citotoxinas/antagonistas & inibidores , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/metabolismo , Leucocidinas/antagonistas & inibidores , Leucocidinas/metabolismo , Neutrófilos/imunologia , Neutrófilos/microbiologia , Organoides/imunologia , Organoides/microbiologia , Organoides/patologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/química
5.
Nat Methods ; 14(5): 521-530, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28369043

RESUMO

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.


Assuntos
Órgãos Artificiais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Organoides/citologia , Linfócitos T/citologia , Timo/citologia , Biotecnologia/métodos , Células-Tronco Hematopoéticas/imunologia , Humanos , Organoides/imunologia , Células-Tronco/citologia , Células-Tronco/imunologia , Linfócitos T/imunologia , Timo/imunologia
7.
Clin Exp Immunol ; 185(3): 338-47, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27084834

RESUMO

Secretory immunoglobulin A (SIgA) antibodies play an important role in protecting the mucosal surfaces against pathogens and maintaining homeostasis with the commensal microbiota. Because a substantial portion of the gut microbiota is coated with SIgA, we hypothesized that microbiota-SIgA complexes are important for the maintenance of gut homeostasis. Here we investigated the relationship between microbiota-SIgA complexes and inflammatory epithelial cell responses. We used a multi-cellular three-dimensional (3D) organotypical model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes/monocytes, endothelial cells and fibroblasts. We also used human SIgA from human colostrum, and a prominent bacterial member of the first colonizers, Escherichia coli, as a surrogate commensal. We found that free and microbiota-complexed SIgA triggered different epithelial responses. While free SIgA up-regulated mucus production, expression of polymeric immunoglobulin receptor (pIgR) and secretion of interleukin-8 and tumoir necrosis factor-α, microbiota-complexed SIgA mitigated these responses. These results suggest that free and complexed SIgA have different functions as immunoregulatory agents in the gut and that an imbalance between the two may affect gut homeostasis.


Assuntos
Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Imunoglobulina A Secretora/química , Imunoglobulina A Secretora/imunologia , Intestinos/imunologia , Organoides/citologia , Organoides/imunologia , Colostro/imunologia , Escherichia coli/imunologia , Escherichia coli/fisiologia , Homeostase , Humanos , Imunidade nas Mucosas/imunologia , Imunoglobulina A Secretora/isolamento & purificação , Imunoglobulina A Secretora/farmacologia , Inflamação , Interleucina-8/metabolismo , Mucosa Intestinal/imunologia , Intestinos/citologia , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Organoides/microbiologia , Fator de Necrose Tumoral alfa/metabolismo
8.
Sci Rep ; 6: 21868, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26902749

RESUMO

Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes.


Assuntos
Células Endoteliais/imunologia , Hepatócitos/imunologia , Fígado/imunologia , Modelos Biológicos , Monócitos/imunologia , Organoides/imunologia , Albuminas/genética , Albuminas/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Apolipoproteínas B/genética , Apolipoproteínas B/imunologia , Caderinas/genética , Caderinas/imunologia , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/imunologia , Comunicação Celular/imunologia , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Inflamação , Dispositivos Lab-On-A-Chip , Lipopolissacarídeos/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Organoides/citologia , Organoides/efeitos dos fármacos , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
9.
Nature ; 528(7583): 560-564, 2015 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-26649819

RESUMO

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.


Assuntos
Células Epiteliais/citologia , Interleucinas/imunologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Regeneração , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Interleucinas/deficiência , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/imunologia , Celulas de Paneth/citologia , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Nicho de Células-Tronco
10.
Biomaterials ; 63: 24-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26072995

RESUMO

Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell-cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ∼100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.


Assuntos
Linfócitos B/citologia , Materiais Biocompatíveis/química , Centro Germinativo/citologia , Nanocompostos/química , Organoides/citologia , Animais , Linfócitos B/imunologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Centro Germinativo/imunologia , Camundongos Endogâmicos C57BL , Nanocompostos/ultraestrutura , Técnicas de Cultura de Órgãos/métodos , Organoides/imunologia , Engenharia Tecidual/métodos
11.
Regen Med ; 10(3): 317-29, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25933240

RESUMO

The thymus is required for generation of a self-tolerant, self-restricted T-cell repertoire. The capacity to manipulate or replace thymus function therapeutically would be beneficial in a variety of clinical settings, including for improving recovery following bone marrow transplantation, restoring immune system function in the elderly and promoting tolerance to transplanted organs or cells. An attractive strategy would be transplantation of thymus organoids generated from cells produced in vitro, for instance from pluripotent stem cells. Here, we review recent progress toward this goal, focusing on advances in directing differentiation of pluripotent stem cells to thymic epithelial cells, a key cell type of the thymic stroma, and related direct reprogramming strategies.


Assuntos
Reprogramação Celular/imunologia , Tolerância Imunológica , Organoides , Células-Tronco Pluripotentes , Nicho de Células-Tronco/imunologia , Timo , Animais , Humanos , Organoides/citologia , Organoides/imunologia , Organoides/transplante , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/imunologia , Timo/citologia , Timo/imunologia , Timo/transplante
12.
Mol Ther ; 23(7): 1262-1277, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25903472

RESUMO

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.


Assuntos
Células Epiteliais/imunologia , Tolerância Imunológica/imunologia , Timo/crescimento & desenvolvimento , Transplante Homólogo , Aloenxertos/imunologia , Animais , Bioengenharia , Células Epiteliais/citologia , Camundongos , Organoides/imunologia , Medicina Regenerativa , Timo/citologia , Timo/imunologia
13.
Oncol Rep ; 32(5): 1820-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25174410

RESUMO

Oral mucositis is a severe complication of radiotherapy. Hence, it may constitute a serious medical safety risk for astronauts during extended space flights, such as missions to Mars, during which they are exposed to heavy-ion irradiation. For risk assessment of developing radiation-induced mucositis, a three-dimensional (3D) organotypic oral mucosa model was irradiated with 12C heavy ions or X­rays. The present study focused mainly on early radiation­induced effects, such as the activation of nuclear factor κB (NFκB) and the expression or release of pro-inflammatory marker molecules. The 3D oral mucosa models with or without peripheral blood mononuclear cells (PBMCs) were irradiated with X­rays or 12C heavy ions followed by snap freezing. Subsequently, cryosections were derived from the specimens, which were immunostained for analysis of compactness, DNA double strand breaks (DSB) and activation of NFκB. Radiation­induced release of interleukin 6 (IL6) and interleukin 8 (IL8) was quantified by ELISA. Quantification of the DNA damage in irradiated mucosa models revealed distinctly more DSB after heavy-ion irradiation compared to X­rays at definite time points, suggesting a higher gene toxicity of heavy ions. NFκB activation was observed after treatment with X­rays or 12C particles. ELISA analyses showed significantly higher IL6 and IL8 levels after irradiation with X­rays and 12C particles compared to non-irradiated controls, whereas co­cultures including PBMCs released 2 to 3-fold higher interleukin concentrations compared to mucosa models without PBMCs. In this study, we demonstrated that several pro-inflammatory markers are induced by X­rays and heavy-ion irradiation within an oral mucosa model. This suggests that oral mucositis indeed poses a risk for astronauts on extended space flights.


Assuntos
Carbono/efeitos adversos , Citocinas/metabolismo , Íons Pesados/efeitos adversos , Mucosa Bucal/patologia , Organoides/efeitos da radiação , Células Cultivadas , Técnicas de Cocultura , Dano ao DNA , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos da radiação , Modelos Biológicos , Mucosa Bucal/imunologia , Mucosa Bucal/efeitos da radiação , Organoides/imunologia , Organoides/patologia , Raios X/efeitos adversos
14.
Am J Physiol Gastrointest Liver Physiol ; 306(7): G582-93, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24503767

RESUMO

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ(-/-)) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ(-/-) mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ(-/-) mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ(-/-) mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ(-/-) and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.


Assuntos
Células Caliciformes/metabolismo , Imunidade nas Mucosas , Intestino Delgado/metabolismo , Mucinas/metabolismo , Muco/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Animais , Antígenos de Superfície/metabolismo , Colite/induzido quimicamente , Colite/imunologia , Colite/metabolismo , Colite/prevenção & controle , Sulfato de Dextrana , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Glicosilação , Células Caliciformes/imunologia , Células Caliciformes/patologia , Homeostase , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/metabolismo , Mucinas/genética , Organoides/imunologia , Organoides/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Fatores de Tempo , Técnicas de Cultura de Tecidos
15.
Biochem Biophys Res Commun ; 438(4): 640-6, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23948691

RESUMO

BACKGROUND: Prostaglandin E2 (PGE2) is a proinflammatory mediator and activates the canonical Wnt-ß-catenin signaling pathway in hematopoietic stem cells. The SZ95 cell line was established from human sebaceous gland cells and is studied as a model system for these cells. Given that 2D culture of SZ95 cells does not recapitulate the organization of sebaceous glands in situ, we developed a 3D culture system for these cells and examined the effects of PGE2 on cell morphology and function. RESULTS: SZ95 cells maintained in 3D culture formed organoids that mimicked the organization of sebaceous glands in situ, including the establishment of a basement membrane. Organoids exposed to PGE2 were larger and adopted a more complex organization compared with control organoids. PGE2 activated the canonical Wnt signaling pathway as well as increased cell viability and proliferation, mitochondrial metabolism, and lipid synthesis in the organoids. CONCLUSIONS: Culture of SZ95 cells in 3D culture system recapitulates the structure and susceptibility to PGE2 of sebaceous glands in situ and should prove useful for studies of the response of these glands to inflammation and other environmental stressors. Our results also implicate PGE2-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of "semi-vivo" sebaceous glands.


Assuntos
Dinoprostona/imunologia , Organoides/citologia , Glândulas Sebáceas/citologia , Via de Sinalização Wnt , Animais , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Metabolismo dos Lipídeos , Lipídeos/imunologia , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Organoides/imunologia , Organoides/metabolismo
16.
Br J Nutr ; 109 Suppl 2: S27-34, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23360878

RESUMO

Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host-microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.


Assuntos
Células Dendríticas/imunologia , Interações Hospedeiro-Parasita , Mucosa Intestinal/imunologia , Modelos Biológicos , Probióticos , Animais , Reatores Biológicos/microbiologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/microbiologia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Macrófagos/citologia , Macrófagos/imunologia , Organoides/imunologia , Organoides/microbiologia , Técnicas de Cultura de Tecidos
17.
J Biotechnol ; 148(1): 38-45, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20416346

RESUMO

Pharmaceutical drugs and compounds used for consumer products may bear the risk of unexpected immuno-toxicological side effects, such as sensitization, allergy, anaphylaxis or immunogenicity. Modern biopharmaceuticals with high potency and target specificity, like antibodies and cytokines need to be tested for their therapeutical doses, their exposition regimens and their immune functionality prior to first-in-man applications. For the latter, existing in vitro tests and animal models do not sufficiently reflect the complexity and specificity of the human immune system. Even novel humanised animal models have limitations in their systemic reactions. Monolayer or suspended cell culture possesses neither tissue functionality nor organ physiology, and also cannot be used for long term culture and experiments. In contrast, solid tissue biopsies, e.g. tonsil preparations of tonsillitis patients typically show inflammatory artefacts and degrade in long term culture due to preparation-induced damage. The construction of tissue-like structures in vitro, so-called "micro-organoids", can overcome these limitations. Key structures of secondary lymphatic organs, e.g. lymph nodes or the spleen are the primary lymphatic follicles and germinal centres, in particular during the "activated state" of an inflammation or infection. To remodel lymphatic follicles, functional and structural cells, e.g. lymphoid cells derived from peripheral blood mononuclear cells (PBMCs) and stromal cells need to be combined with biogenic or artificial matrices and scaffolds to produce a suitable 3D tissue-mimicking environment. Therefore, a unique human lymph node model (HuALN) was designed to operate over several weeks, and allow long term and repeated drug exposure to induce and monitor both cellular and humoral immune responses. Cellular immunity is monitored, for example, by cytokine release patterns; humoral immunity is analysed, for example, by B cell activation, plasma cell formation and antibody secretion profiles (IgM and IgG). Moreover, cellular composition and micro-organoid formation are analysed by flow cytometry, histology and in situ imaging.


Assuntos
Reatores Biológicos , Linfonodos , Modelos Imunológicos , Organoides , Técnicas de Cultura de Tecidos , Citocinas/análise , Citocinas/metabolismo , Histocitoquímica , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina M/análise , Imunoglobulina M/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Organoides/imunologia , Organoides/metabolismo , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos
18.
Methods Mol Biol ; 380: 163-70, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17876092

RESUMO

The thymic microenvironment provides essential support for the generation of a functional and diverse population of human T cells. In particular, the three-dimensional (3D) thymic architecture contributes to critical cell-cell interactions. We report that thymic stroma, arrayed on a synthetic 3D matrix, supports the development of functional human T cells from hematopoietic precursor cells. Newly generated T cells contain T-cell receptor excision circles and are both fully mature and functional. The coculture of T-cell progenitors with thymic stroma can thus be used to generate de novo functional and diverse T-cell populations. This novel tissue engineered thymic system has biological applications for the study of T-lymphopoiesis and self-tolerance as well as potential therapeutic applications including the immune reconstitution of immunocompromised patients and the induction of tolerance in individuals receiving tissue or organ transplants.


Assuntos
Organoides , Timo , Engenharia Tecidual , Animais , Humanos , Organoides/citologia , Organoides/imunologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia
19.
Nat Biotechnol ; 22(12): 1539-45, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15568019

RESUMO

Stromal cells play an important role in the formation of the normal organized microarchitecture of secondary lymphoid organs. Here we demonstrate that a tissue-engineered, lymphoid tissue-like organoid, which was constructed by transplantation of stromal cells embedded in biocompatible scaffolds into the renal subcapsular space in mice, had an organized tissue structure similar to secondary lymphoid organs. This organoid contained compartmentalized B-cell and T-cell clusters, high endothelial venule-like vessels, germinal centers and follicular dendritic cell networks. Furthermore, the organoid was transplantable to naive normal or severe combined immunodeficiency (SCID) mice, and antigen-specific, IgG-isotype antibody formation could be induced soon after intravenous administration of the antigen. This simplified system of lymphoid tissue-like organoid construction will facilitate analyses of cell-cell interactions required for development of secondary lymphoid organs and efficient induction of adaptive immune responses, and may have possible applications in the treatment of immune deficiency.


Assuntos
Linfonodos/citologia , Linfócitos/citologia , Organoides/citologia , Timo/citologia , Timo/transplante , Engenharia Tecidual/métodos , Animais , Bioprótese , Diferenciação Celular , Linhagem Celular , Estudos de Viabilidade , Linfonodos/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Organoides/imunologia , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/transplante , Timo/imunologia
20.
Br J Cancer ; 77(5): 753-9, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9514054

RESUMO

The high linear energy transfer, alpha-particle-emitting radionuclide astatine-211 (211At) is of interest for certain therapeutic applications; however, because of the 55- to 70-microm path length of its alpha-particles, achieving homogeneous tracer distribution is critical. Hyperthermia may enhance the therapeutic efficacy of alpha-particle endoradiotherapy if it can improve tracer distribution. In this study, we have investigated whether hyperthermia increased the cytotoxicity of an 211At-labelled monoclonal antibody (MAb) in tumour spheroids with a radius (approximately 100 microm) greater than the range of 211At alpha-particles. Hyperthermia for 1 h at 42 degrees C was used because this treatment itself resulted in no regrowth delay. Radiolabelled chimeric MAb 81C6 reactive with the extracellular matrix antigen tenascin was added to spheroids grown from the D-247 MG human glioma cell line at activity concentrations ranging from 0.125 to 250 kBq ml(-1). A significant regrowth delay was observed at 125 and 250 kBq ml(-1) in both hyperthermia-treated and untreated spheroids. For groups receiving hyperthermia, no increase in cytotoxicity was seen compared with normothermic controls at any activity concentration. These results and those from autoradiographs indicate that hyperthermia at 42 degrees C for 1 h had no significant effect on the uptake or distribution of this antitenascin MAb in D-247 MG spheroids.


Assuntos
Partículas alfa , Anticorpos Monoclonais/uso terapêutico , Astato/uso terapêutico , Gliossarcoma/patologia , Organoides/efeitos da radiação , Radioimunoterapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Transporte Biológico , Humanos , Hipertermia Induzida , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina G/uso terapêutico , Camundongos , Organoides/imunologia , Organoides/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Tenascina/imunologia , Células Tumorais Cultivadas/efeitos da radiação
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