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1.
Blood ; 137(4): 500-512, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33507291

RESUMO

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

2.
Cell ; 183(5): 1219-1233.e18, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33242418

RESUMO

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

3.
FASEB J ; 34(9): 12963-12975, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32772418

RESUMO

Immunotherapy is a powerful treatment strategy being applied to cancer, autoimmune diseases, allergies, and transplantation. Although therapeutic monoclonal antibodies (mAbs) have demonstrated significant clinical efficacy, there is also the potential for severe adverse events, including cytokine release syndrome (CRS). CRS is characterized by the rapid production of inflammatory cytokines following delivery of therapy, with symptoms ranging from mild fever to life-threating pathology and multi-organ failure. Overall there is a paucity of models to reliably and accurately predict the induction of CRS by immune therapeutics. Here, we describe the development of a humanized mouse model based on the NOD-scid IL2rgnull (NSG) mouse to study CRS in vivo. PBMC-engrafted NSG, NSG-MHC-DKO, and NSG-SGM3 mice were used to study cytokine release in response to treatment with mAb immunotherapies. Our data show that therapeutic-stimulated cytokine release in these PBMC-based NSG models captures the variation in cytokine release between individual donors, is drug dependent, occurs in the absence of acute xeno-GVHD, highlighting the specificity of the assay, and shows a robust response following treatment with a TGN1412 analog, a CD28 superagonist. Overall our results demonstrate that PBMC-engrafted NSG models are rapid, sensitive, and reproducible platforms to screen novel therapeutics for CRS.

4.
Blood ; 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32756933

RESUMO

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia (ALL) where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, shRNA screen in murine T cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cAMP signaling and are under-expressed in GC resistant or relapsed ALL patients. Silencing of the cAMP activating guanine nucleotide binding protein, alpha stimulating Gnas gene, interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that Prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC re-sensitization in relapsed pediatric T-ALL.

5.
Cell Rep ; 32(2): 107894, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32668238

RESUMO

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes, we differentiated human induced pluripotent stem cells into pancreatic endocrine cells, including ß cells. Here, we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived ß cells, along with a reduced effect on α cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response.

6.
Endocrinology ; 161(8)2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428240

RESUMO

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.

7.
Nat Biomed Eng ; 4(8): 814-826, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32231313

RESUMO

The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes <0.8 µm prevented their infiltration by immune cells. A synthetic polymer coating prevented fibrosis and was necessary for the long-term function of the device. For >130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression.


Assuntos
Transplante de Células/métodos , Sobrevivência de Enxerto , Próteses e Implantes , Animais , Cápsulas , Transplante de Células/instrumentação , Materiais Revestidos Biocompatíveis , Diabetes Mellitus Experimental/terapia , Desenho de Equipamento , Eritropoetina/genética , Eritropoetina/metabolismo , Reação a Corpo Estranho/prevenção & controle , Células HEK293 , Humanos , Ilhotas Pancreáticas , Transplante das Ilhotas Pancreáticas/instrumentação , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Permeabilidade , Ratos , Transplante Heterólogo
8.
Microorganisms ; 8(2)2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32093375

RESUMO

Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant ß-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived ß (SC-ß) cells are insulin-producing cell clusters that closely resemble native ß cells. To better understand the events precipitated by enteroviral infection of ß cells, we investigated transcriptional and proteomic changes in SC-ß cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-ß cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-ß cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-ß cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-ß cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.

9.
FASEB J ; 34(1): 1901-1911, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914605

RESUMO

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.


Assuntos
Células Endócrinas/fisiologia , Ilhotas Pancreáticas/fisiologia , Transcriptoma/fisiologia , Adulto , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Células Endócrinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Sobrevivência de Enxerto/fisiologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Camundongos , Transplante Heterólogo/métodos , Adulto Jovem
10.
JCI Insight ; 5(1)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31941840

RESUMO

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of ß cell mass. Interestingly, these ß cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced ß cell dysfunction and partially prevented SIR-induced ß cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human ß cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.

11.
Int Arch Allergy Immunol ; 180(2): 91-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31401630

RESUMO

INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.


Assuntos
Anafilaxia/prevenção & controle , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Eosinófilos/imunologia , Lectinas/imunologia , Mastócitos/imunologia , Anafilaxia/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Basófilos/imunologia , Humanos , Camundongos , Ácido N-Acetilneuramínico/imunologia , Receptores de IgG/imunologia
12.
Cell Host Microbe ; 26(3): 426-434.e6, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31447308

RESUMO

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Aminoácidos Aromáticos/biossíntese , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Ilhas Genômicas/genética , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Ferro/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Obesos , Camundongos SCID , Salmonella typhi/crescimento & desenvolvimento , Sideróforos/metabolismo , Células THP-1/microbiologia , Febre Tifoide , Virulência/genética
13.
Nat Mater ; 18(8): 892-904, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31235902

RESUMO

Implantable medical devices have revolutionized modern medicine. However, immune-mediated foreign body response (FBR) to the materials of these devices can limit their function or even induce failure. Here we describe long-term controlled-release formulations for local anti-inflammatory release through the development of compact, solvent-free crystals. The compact lattice structure of these crystals allows for very slow, surface dissolution and high drug density. These formulations suppress FBR in both rodents and non-human primates for at least 1.3 years and 6 months, respectively. Formulations inhibited fibrosis across multiple implant sites-subcutaneous, intraperitoneal and intramuscular. In particular, incorporation of GW2580, a colony stimulating factor 1 receptor inhibitor, into a range of devices, including human islet microencapsulation systems, electrode-based continuous glucose-sensing monitors and muscle-stimulating devices, inhibits fibrosis, thereby allowing for extended function. We believe that local, long-term controlled release with the crystal formulations described here enhances and extends function in a range of medical devices and provides a generalized solution to the local immune response to implanted biomaterials.


Assuntos
Fibrose/etiologia , Fibrose/prevenção & controle , Próteses e Implantes/efeitos adversos , Animais , Preparações de Ação Retardada , Composição de Medicamentos , Macrófagos/efeitos dos fármacos , Roedores
14.
Mamm Genome ; 30(5-6): 123-142, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30847553

RESUMO

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic.


Assuntos
Modelos Animais de Doenças , Doenças do Sistema Imunitário/imunologia , Medicina de Precisão , Animais , Transplante de Células , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/fisiologia , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/patologia , Camundongos , Transplante Heterólogo
15.
J Immunol ; 202(3): 799-804, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30593536

RESUMO

Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.


Assuntos
Anticorpos Monoclonais Humanizados/genética , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Testes de Neutralização
16.
Cell Metab ; 29(3): 745-754.e4, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30449685

RESUMO

Identification of cell-surface markers specific to human pancreatic ß cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human ß cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet ß cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human ß cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human ß cells. Thus, NTPDase3 is a cell-surface biomarker of adult human ß cells, and the antibody directed to this protein should be a useful new reagent for ß cell sorting, in vivo imaging, and targeting.


Assuntos
Adenosina Trifosfatases/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Adulto , Animais , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/patologia , Adulto Jovem
17.
FASEB J ; 33(3): 3137-3151, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30383447

RESUMO

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Linfócitos T/imunologia , Animais , Feminino , Genes MHC Classe I , Genes MHC da Classe II , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo
18.
EBioMedicine ; 38: 79-88, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30497977

RESUMO

BACKGROUND: Nanomedicine offers an excellent opportunity to tackle treatment-refractory malignancies by enhancing the delivery of therapeutics to the tumor site. High endothelial venules (HEVs) are found primarily in lymph nodes or formed de novo in peripheral tissues during inflammatory responses. They express peripheral node addressin (PNAd), which is recognized by the monoclonal antibody MECA79. METHODS: Here, we demonstrated that HEVs form de novo in human pancreatic ductal adenocarcinoma (PDAC). We engineered MECA79 coated nanoparticles (MECA79-NPs) that recognize these ectopic HEVs in PDAC. FINDINGS: The trafficking of MECA79-NPs following intravenous delivery to human PDAC implanted in a humanized mouse model was more robust than non-conjugated NPs. Treatment with MECA79-Taxol-NPs augmented the delivery of Paclitaxel (Taxol) to the tumor site and significantly reduced the tumor size. This effect was associated with a higher apoptosis rate of PDAC cells and reduced vascularization within the tumor. INTERPRETATION: Targeting the HEVs of PDAC using MECA79-NPs could lay the ground for the localized delivery of a wide variety of drugs including chemotherapeutic agents. FUND: National Institutes of Health (NIH) grants: T32-EB016652 (B·B.), NIH Cancer Core Grant CA034196 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.).


Assuntos
Carcinoma Ductal Pancreático/patologia , Endotélio Linfático/patologia , Linfonodos/patologia , Neovascularização Patológica , Neoplasias Pancreáticas/patologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Biomarcadores , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Terapia de Alvo Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Neovascularização Patológica/tratamento farmacológico , Paclitaxel/administração & dosagem , Paclitaxel/química , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Nanomedicina Teranóstica , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Transplantation ; 102(8): 1223-1229, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29781950

RESUMO

Beta cell replacement has the potential to restore euglycemia in patients with insulin-dependent diabetes. Although great progress has been made in establishing allogeneic islet transplantation from deceased donors as the standard of care for those with the most labile diabetes, it is also clear that the deceased donor organ supply cannot possibly treat all those who could benefit from restoration of a normal beta cell mass, especially if immunosuppression were not required. Against this background, the International Pancreas and Islet Transplant Association in collaboration with the Harvard Stem Cell Institute, the Juvenile Diabetes Research Foundation (JDRF), and the Helmsley Foundation held a 2-day Key Opinion Leaders Meeting in Boston in 2016 to bring together experts in generating and transplanting beta cells derived from stem cells. The following summary highlights current technology, recent significant breakthroughs, unmet needs and roadblocks to stem cell-derived beta cell therapies, with the aim of spurring future preclinical collaborative investigations and progress toward the clinical application of stem cell-derived beta cells.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/citologia , Transplante de Células-Tronco/métodos , Animais , Boston , Diferenciação Celular , Congressos como Assunto , Edição de Genes , Humanos , Tolerância Imunológica , Células Secretoras de Insulina/imunologia , Transplante das Ilhotas Pancreáticas , Pâncreas/citologia , Transplante de Pâncreas/métodos , Células-Tronco Pluripotentes/citologia , Doadores de Tecidos
20.
Cell Rep ; 22(10): 2667-2676, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514095

RESUMO

Many patients with type 1 diabetes (T1D) have residual ß cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual ß cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant ß cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and ß cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.


Assuntos
Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica , Células Secretoras de Glucagon/metabolismo , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Reprogramação Celular , Criança , Feminino , Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Fenótipo , Doadores de Tecidos , Fatores de Transcrição/metabolismo , Adulto Jovem
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