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1.
Int Arch Allergy Immunol ; : 1-12, 2019 Aug 09.
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.

2.
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.

3.
Nat Mater ; 18(8): 892-904, 2019 Aug.
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.

4.
Mamm Genome ; 30(5-6): 123-142, 2019 Jun.
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.

5.
J Immunol ; 202(3): 799-804, 2019 Feb 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.

6.
EBioMedicine ; 2018 Nov 26.
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 CA034194 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.).

7.
FASEB J ; : fj201800636R, 2018 Nov 01.
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.

8.
Cell Metab ; 2018 Nov 12.
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.

9.
Transplantation ; 102(8): 1223-1229, 2018 Aug.
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.

10.
Cell Rep ; 22(10): 2667-2676, 2018 Mar 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.

11.
FASEB J ; 32(3): 1537-1549, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29146734

RESUMO

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.-Wang, M., Yao, L.-C., Cheng, M., Cai, D., Martinek, J., Pan, C.-X., Shi, W., Ma, A.-H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular/efeitos dos fármacos , Imunoterapia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/imunologia , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Mol Ther ; 25(11): 2477-2489, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29032169

RESUMO

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Hepatócitos/transplante , Transgenes , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Animais , Dependovirus/metabolismo , Modelos Animais de Doenças , Edição de Genes , Expressão Gênica , Inativação Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Sobrevivência de Enxerto , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Transplante Heterólogo , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/enzimologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
13.
J Clin Invest ; 127(10): 3835-3844, 2017 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-28920919

RESUMO

Inadequate pancreatic ß cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human ß cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of ß cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human ß cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human ß cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet ß cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human ß cell proliferation, and identify elements that could be adapted for therapeutic expansion of human ß cells.


Assuntos
Envelhecimento/metabolismo , Calcineurina/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Transdução de Sinais , Adulto , Animais , Ciclina A1/metabolismo , Exenatida , Feminino , Proteína Forkhead Box M1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Insulina/metabolismo , Masculino , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologia
14.
Cell Rep ; 20(8): 1978-1990, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28834758

RESUMO

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Inata/imunologia , Células-Tronco Pluripotentes/metabolismo , Condicionamento Pré-Transplante/métodos , Animais , Modelos Animais de Doenças , Rejeição de Enxerto , Humanos , Camundongos
15.
PLoS One ; 12(6): e0178641, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28605395

RESUMO

BACKGROUND/AIMS: Studies of human cadaveric pancreas specimens indicate that pancreas inflammation plays an important role in type 1 diabetes pathogenesis. Due to the inaccessibility of pancreas in living patients, imaging technology to visualize pancreas inflammation is much in need. In this study, we investigated the feasibility of utilizing ultrasound imaging to assess pancreas inflammation longitudinally in living rats during the progression leading to type 1 diabetes onset. METHODS: The virus-inducible BBDR type 1 diabetes rat model was used to systematically investigate pancreas changes that occur prior to and during development of autoimmunity. The nearly 100% diabetes incidence upon virus induction and the highly consistent time course of this rat model make longitudinal imaging examination possible. A combination of histology, immunoblotting, flow cytometry, and ultrasound imaging technology was used to identify stage-specific pancreas changes. RESULTS: Our histology data indicated that exocrine pancreas tissue of the diabetes-induced rats underwent dramatic changes, including blood vessel dilation and increased CD8+ cell infiltration, at a very early stage of disease initiation. Ultrasound imaging data revealed significant acute and persistent pancreas inflammation in the diabetes-induced rats. The pancreas micro-vasculature was significantly dilated one day after diabetes induction, and large blood vessel (superior mesenteric artery in this study) dilation and inflammation occurred several days later, but still prior to any observable autoimmune cell infiltration of the pancreatic islets. CONCLUSIONS: Our data demonstrate that ultrasound imaging technology can detect pancreas inflammation in living rats during the development of type 1 diabetes. Due to ultrasound's established use as a non-invasive diagnostic tool, it may prove useful in a clinical setting for type 1 diabetes risk prediction prior to autoimmunity and to assess the effectiveness of potential therapeutics.


Assuntos
Diabetes Mellitus Tipo 1/diagnóstico por imagem , Diabetes Mellitus Tipo 1/patologia , Pancreatite/diagnóstico por imagem , Pancreatite/patologia , Ultrassonografia , Animais , Apoptose , Resistência Capilar , Caspase 3/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/etiologia , Modelos Animais de Doenças , Humanos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Microvasos , Pâncreas/irrigação sanguínea , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/complicações , Pancreatite/metabolismo , Prognóstico , Ratos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Ultrassonografia/métodos
16.
J Clin Invest ; 127(6): 2433-2437, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28481220

RESUMO

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.


Assuntos
Movimento Celular , Células-Tronco Hematopoéticas/fisiologia , RNA Mensageiro/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Selectina E , Fucosiltransferases/fisiologia , Glicosilação , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos Endogâmicos NOD , Camundongos SCID , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética
17.
J Immunol Methods ; 446: 47-53, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28390927

RESUMO

Monoclonal antibodies (mAbs) have emerged as a mainstream therapeutic option against cancer. mAbs mediate tumor cell-killing through several mechanisms including complement-dependent cytotoxicity (CDC). However, studies of mAb-mediated CDC against tumor cells remain largely dependent on in vitro systems. Previously developed and widely used NOD-scid IL2rγnull (NSG) mice support enhanced engraftment of many primary human tumors. However, NSG mice have a 2-bp deletion in the coding region of the hemolytic complement (Hc) gene, and it is not possible to evaluate CDC activity in NSG mice. To address this limitation, we generated a novel strain of NSG mice-NSG-Hc1-that have an intact complement system able to generate the membrane attack complex. Utilizing the Daudi Burkitt's human lymphoma cell line, and the anti-human CD20 mAb rituximab, we further demonstrated that the complement system in NSG-Hc1 mice is fully functional. NSG-Hc1 mice expressed CDC activity against Daudi cells in vivo following rituximab treatment and showed longer overall survival compared with rituximab-treated NSG mice that lack hemolytic complement. Our results validate the NSG-Hc1 mouse model as a platform for testing mechanisms underlying CDC in vivo and suggest its potential use to compare complement-dependent and complement-independent cytotoxic activity mediated by therapeutic mAbs.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Proteínas do Sistema Complemento/imunologia , Imunoterapia/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos/administração & dosagem , Antígenos CD20 , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Rituximab
18.
Nat Mater ; 16(6): 671-680, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28319612

RESUMO

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

19.
Stem Cell Res Ther ; 8(1): 65, 2017 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-28283030

RESUMO

BACKGROUND: Poor bone quality, increased fracture risks, and impaired bone healing are orthopedic comorbidities of type 1 diabetes (T1DM). Standard osteogenic growth factor treatments are inadequate in fully rescuing retarded healing of traumatic T1DM long bone injuries where both periosteal and bone marrow niches are disrupted. We test the hypotheses that osteogenesis of bone marrow-derived stromal cells (BMSCs) and periosteum-derived cells (PDCs), two critical skeletal progenitors in long bone healing, are both impaired in T1DM and that they respond differentially to osteogenic bone morphogenetic proteins (BMPs) and/or insulin-like growth factor-1 (IGF-1) rescue. METHODS: BMSCs and PDCs were isolated from Biobreeding Diabetes Prone/Worcester rats acquiring T1DM and normal Wistar rats. Proliferation, osteogenesis, and adipogenesis of the diabetic progenitors were compared with normal controls. Responses of diabetic progenitors to osteogenesis rescue by rhBMP-2/7 heterodimer (45 or 300 ng/ml) and/or rhIGF-1 (15 or 100 ng/ml) in normal and high glucose cultures were examined by alizarin red staining and qPCR. RESULTS: Diabetic BMSCs and PDCs proliferated slower and underwent poorer osteogenesis than nondiabetic controls, and these impairments were exacerbated in high glucose cultures. Osteogenesis of diabetic PDCs was rescued by rhBMP-2/7 or rhBMP-2/7 + rhIGF-1 in both normal and high glucose cultures in a dose-dependent manner. Diabetic BMSCs, however, only responded to 300 ng/nl rhBMP-2/7 with/without 100 ng/ml rhIGF-1 in normal but not high glucose osteogenic culture. IGF-1 alone was insufficient in rescuing the osteogenesis of either diabetic progenitor. Supplementing rhBMP-2/7 in high glucose osteogenic culture significantly enhanced gene expressions of type 1 collagen (Col 1), osteocalcin (OCN), and glucose transporter 1 (GLUT1) while suppressing that of adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) in diabetic PDCs. The same treatment in high glucose culture only resulted in a moderate increase in Col 1, but no significant changes in OCN or GLUT1 expressions in diabetic BMSCs. CONCLUSIONS: This study demonstrates more effective osteogenesis rescue of diabetic PDCs than BMSCs by rhBMP-2/7 with/without rhIGF-1 in a hyperglycemia environment, underscoring the necessity to tailor biochemical therapeutics to specific skeletal progenitor niches. Our data also suggest potential benefits of combining growth factor treatment with blood glucose management to optimize orthopedic therapeutic outcomes for T1DM patients.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 7/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fator de Crescimento Insulin-Like I/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/agonistas , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Periósteo/efeitos dos fármacos , Periósteo/metabolismo , Periósteo/patologia , Cultura Primária de Células , Ratos , Ratos Endogâmicos BB , Ratos Wistar , Proteínas Recombinantes/farmacologia
20.
Annu Rev Pathol ; 12: 187-215, 2017 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-27959627

RESUMO

Immunodeficient mice engrafted with functional human cells and tissues, that is, humanized mice, have become increasingly important as small, preclinical animal models for the study of human diseases. Since the description of immunodeficient mice bearing mutations in the IL2 receptor common gamma chain (IL2rgnull) in the early 2000s, investigators have been able to engraft murine recipients with human hematopoietic stem cells that develop into functional human immune systems. These mice can also be engrafted with human tissues such as islets, liver, skin, and most solid and hematologic cancers. Humanized mice are permitting significant progress in studies of human infectious disease, cancer, regenerative medicine, graft-versus-host disease, allergies, and immunity. Ultimately, use of humanized mice may lead to the implementation of truly personalized medicine in the clinic. This review discusses recent progress in the development and use of humanized mice and highlights their utility for the study of human diseases.


Assuntos
Doenças Transmissíveis/terapia , Modelos Animais de Doenças , Sistema Imunitário/imunologia , Animais , Doenças Transmissíveis/imunologia , Humanos , Camundongos , Camundongos SCID
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