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1.
J Health Care Poor Underserved ; 32(2): 598-606, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120960

RESUMO

In response to the COVID-19 pandemic, the Herbert Wertheim College of Medicine's Neighborhood Health Education Learning Program (NeighborhoodHELP) initiated a longitudinal assessment and mitigation of social and health care challenges for a population of approximately 850 underserved households. Here, we describe the needs assessment, ensuing interventions, and lessons learned during this pandemic.


Assuntos
COVID-19/epidemiologia , Determinação de Necessidades de Cuidados de Saúde , Populações Vulneráveis , Adolescente , Adulto , Idoso , Criança , Serviços de Saúde Comunitária , Informação de Saúde ao Consumidor , Feminino , Florida/epidemiologia , Assistência Alimentar , Necessidades e Demandas de Serviços de Saúde , Humanos , Masculino , Área Carente de Assistência Médica , Pessoa de Meia-Idade , Pandemias , Determinantes Sociais da Saúde , Adulto Jovem
2.
J Histochem Cytochem ; : 221554211025482, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34165342

RESUMO

Brain tumors in adults may be infrequent when compared with other cancer etiologies, but they remain one of the deadliest with bleak survival rates. Current treatment modalities encompass surgical resection, chemotherapy, and radiotherapy. However, increasing resistance rates are being witnessed, and this has been attributed, in part, to cancer stem cells (CSCs). CSCs are a subpopulation of cancer cells that reside within the tumor bulk and have the capacity for self-renewal and can differentiate and proliferate into multiple cell lineages. Studying those CSCs enables an increasing understanding of carcinogenesis, and targeting CSCs may overcome existing treatment resistance. One approach to weaponize new drugs is to target these CSCs through drug repurposing which entails using drugs, which are Food and Drug Administration-approved and safe for one defined disease, for a new indication. This approach serves to save both time and money that would otherwise be spent in designing a totally new therapy. In this review, we will illustrate drug repurposing strategies that have been used in brain tumors and then further elaborate on how these approaches, specifically those that target the resident CSCs, can help take the field of drug repurposing to a new level.

3.
FEBS J ; 2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-33934527

RESUMO

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as "tumor-associated carbohydrate antigens". Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

4.
FEBS J ; 288(16): 4746-4772, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33752265

RESUMO

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell-cell communications and in the regulation of immune responses. Through the interaction with glycan-binding receptors, glycans are able to affect the activation status of antigen-presenting cells, leading either to induction of pro-inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco-chemists and glyco-immunologists to develop glycan-based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan-modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen-presenting cells, like dendritic cells. In addition, we address how glycan-based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan-based therapies, including chimeric antigen receptor (CAR)-T cells to achieve targeting of tumor-associated glycan-specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity.


Assuntos
Autoimunidade/imunologia , Polissacarídeos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular/imunologia , Humanos , Nanopartículas/química , Polissacarídeos/química , Processamento de Proteína Pós-Traducional
5.
Chem Commun (Camb) ; 57(9): 1145-1148, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33411866

RESUMO

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic of l-fucose that markedly inhibits the creation of sLeX by FTVI and FTVII, but has no effect on creation of LeX by FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.


Assuntos
Fucose/análogos & derivados , Fucose/farmacologia , Fucosiltransferases/antagonistas & inibidores , Linhagem Celular Tumoral , Fucose/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras
6.
Front Cell Dev Biol ; 8: 584074, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33324641

RESUMO

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

8.
J Immunol ; 205(7): 1920-1932, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32868410

RESUMO

Sialyl Lewis X (sLeX) regulates T cell trafficking from the vasculature into skin and sites of inflammation, thereby playing a critical role in immunity. In healthy persons, only a small proportion of human blood T cells express sLeX, and their function is not fully defined. Using a combination of biochemical and functional studies, we find that human blood sLeX+CD4+T cells comprise a subpopulation expressing high levels of Th2 and Th17 cytokines, chemokine receptors CCR4 and CCR6, and the transcription factors GATA-3 and RORγT. Additionally, sLeX+CD4+T cells exclusively contain the regulatory T cell population (CD127lowCD25high and FOXP3+) and characteristically display immune-suppressive molecules, including the coinhibitor receptors PD-1 and CTLA-4. Among CD8+T cells, sLeX expression distinguishes a subset displaying low expression of cytotoxic effector molecules, perforin and granzyme ß, with reduced degranulation and CD57 expression and, consistently, marginal cytolytic capacity after TCR engagement. Furthermore, sLeX+CD8+T cells present a pattern of features consistent with Th cell-like phenotype, including release of pertinent Tc2 cytokines and elevated expression of CD40L. Together, these findings reveal that sLeX display is associated with unique functional specialization of both CD4+ and CD8+T cells and indicate that circulating T cells that are primed to migrate to lesional sites at onset of inflammation are not poised for cytotoxic function.


Assuntos
Antígeno Sialil Lewis X/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Tolerância Central , Citotoxicidade Imunológica , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Memória Imunológica , Ativação Linfocitária , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tolerância Periférica , Receptor de Morte Celular Programada 1/metabolismo , Antígeno Sialil Lewis X/genética
9.
Sci Rep ; 10(1): 13162, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753748

RESUMO

A common missense variant in SLC39A8 is convincingly associated with schizophrenia and several additional phenotypes. Homozygous loss-of-function mutations in SLC39A8 result in undetectable serum manganese (Mn) and a Congenital Disorder of Glycosylation (CDG) due to the exquisite sensitivity of glycosyltransferases to Mn concentration. Here, we identified several Mn-related changes in human carriers of the common SLC39A8 missense allele. Analysis of structural brain MRI scans showed a dose-dependent change in the ratio of T2w to T1w signal in several regions. Comprehensive trace element analysis confirmed a specific reduction of only serum Mn, and plasma protein N-glycome profiling revealed reduced complexity and branching. N-glycome profiling from two individuals with SLC39A8-CDG showed similar but more severe alterations in branching that improved with Mn supplementation, suggesting that the common variant exists on a spectrum of hypofunction with potential for reversibility. Characterizing the functional impact of this variant will enhance our understanding of schizophrenia pathogenesis and identify novel therapeutic targets and biomarkers.


Assuntos
Encéfalo/diagnóstico por imagem , Proteínas de Transporte de Cátions/genética , Manganês/metabolismo , Esquizofrenia/genética , Encéfalo/metabolismo , Feminino , Glicosilação , Humanos , Mutação com Perda de Função , Imageamento por Ressonância Magnética , Masculino , Manganês/sangue , Mutação de Sentido Incorreto , Polissacarídeos/sangue , Esquizofrenia/diagnóstico por imagem , Esquizofrenia/metabolismo
10.
J Cell Mol Med ; 24(14): 8031-8044, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32519822

RESUMO

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapy for the treatment of high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. Acute graft-versus-host disease (aGvHD) remains the most frequent cause of non-relapse mortality following allo-HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug successfully employed for prophylaxis and treatment of veno-occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD-associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte-endothelial interactions by down-regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches.


Assuntos
Comunicação Celular/efeitos dos fármacos , Endotélio/metabolismo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Leucócitos/metabolismo , Polidesoxirribonucleotídeos/farmacologia , Doença Aguda , Animais , Biomarcadores , Biópsia , Comunicação Celular/imunologia , Linhagem Celular , Quimiotaxia de Leucócito/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Camundongos , Doadores de Tecidos , Transplante Homólogo
11.
Acad Med ; 95(9S A Snapshot of Medical Student Education in the United States and Canada: Reports From 145 Schools): S103-S106, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33626657
12.
J Biol Chem ; 294(48): 18465-18474, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31628196

RESUMO

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLe X ), displayed on circulating cells. Although chimeric antigen receptor (CAR) T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLe X and do not bind E-selectin. However, enforced sLe X display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom installation of sLe X programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.


Assuntos
Selectina E/metabolismo , Glicoproteínas/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Antígeno Sialil Lewis X/metabolismo , Linfócitos T/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Células Cultivadas , Fucose/metabolismo , Glicosilação , Humanos , Imunoterapia Adotiva/métodos , Ligantes , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Ligação Proteica , Engenharia de Proteínas/métodos
13.
Front Immunol ; 10: 1151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31231366

RESUMO

Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.

14.
Nat Cell Biol ; 21(5): 627-639, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988423

RESUMO

How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial-mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal-epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal-epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal-epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.


Assuntos
Neoplasias Ósseas/genética , Selectina E/genética , Fucosiltransferases/genética , Metástase Neoplásica/genética , Neoplasias/genética , Animais , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Camundongos , Metástase Neoplásica/patologia , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sialoglicoproteínas/genética , Transdução de Sinais/genética , Nicho de Células-Tronco/genética , Ativação Transcricional/genética , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cytotherapy ; 20(9): 1110-1123, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30170815

RESUMO

BACKGROUND: The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. METHODS: BM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. RESULTS: Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DISCUSSION: The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.


Assuntos
Biotecnologia/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Biotecnologia/normas , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Criopreservação , Selectina E/metabolismo , Células Endoteliais/metabolismo , Fucose/metabolismo , Fucosiltransferases/metabolismo , Glicosilação , Humanos , Receptores de Hialuronatos/metabolismo , Imunofenotipagem , Transcriptoma
17.
J Immunol ; 201(3): 1030-1043, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29941663

RESUMO

The success of dendritic cell (DC)-based immunotherapeutics critically hinges on the capacity of the vascularly administered cells to enter tissues. Transendothelial migration (TEM) is dictated by an ordered cascade of receptor/ligand interactions. In this study, we examined the key molecular effectors of TEM of human monocyte-derived DCs (mo-DCs) generated by clinically relevant methods: CD14 selection (CD14-S) and plastic adherence selection (PA-S). Without chemokine input, CD14-S cells undergo greater TEM than PA-S cells over TNF-α-stimulated HUVECs. TEM of CD14-S mo-DCs is E-selectin/very late Ag-4 (VLA-4) dependent, and engagement of E-selectin ligands activates VLA-4 on CD14-S mo-DCs but not on PA-S mo-DCs. E-selectin binding glycoforms of P-selectin glycoprotein ligand-1 (PSGL-1) (i.e., cutaneous lymphocyte Ag [CLA]) and CD44 (i.e., hematopoietic cell E-selectin/L-selectin ligand [HCELL]) are both expressed on CD14-S mo-DCs, but only CLA is expressed on PA-S mo-DCs. To elucidate the effect of CD44 or PSGL-1 engagement, mo-DCs were pretreated with their ligands. Ligation of CD44 on CD14-S mo-DCs triggers VLA-4 activation and TEM, whereas PSGL-1 ligation does not. HCELL expression on CD14-S mo-DC can be enforced by cell surface exofucosylation, yielding increased TEM in vitro and enhanced extravasation into bone marrow in vivo. These findings highlight structural and functional pleiotropism of CD44 in priming TEM of mo-DCs and suggest that strategies to enforce HCELL expression may boost TEM of systemically administered CD14-S mo-DCs.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Receptores de Hialuronatos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular/fisiologia , Quimiocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Receptores de Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Fator de Necrose Tumoral alfa/metabolismo
18.
BMC Cancer ; 18(1): 495, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29716546

RESUMO

BACKGROUND: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. METHODS: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. RESULTS: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. CONCLUSIONS: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.


Assuntos
Selectina E/metabolismo , Ligantes , Neoplasias/metabolismo , Biomarcadores , Selectina E/genética , Humanos , Imuno-Histoquímica , Neoplasias/genética , Neoplasias/patologia , Inclusão em Parafina
19.
J Biol Chem ; 293(19): 7300-7314, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29593094

RESUMO

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (LeX; CD15) and/or sialyl Lewis-X (sLeX; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human LeX/sLeX biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLeX is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize LeX, and, among all FTs, FT6 holds a unique capacity in creating sLeX and LeX determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLeX on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes.


Assuntos
Fucosiltransferases/metabolismo , Isoenzimas/metabolismo , Antígenos CD15/metabolismo , Células-Tronco Mesenquimais/enzimologia , Amino Açúcares/metabolismo , Citometria de Fluxo , Fucosiltransferases/genética , Glicoconjugados/metabolismo , Glicômica , Humanos , Isoenzimas/genética , Antígenos CD15/genética , Espectrometria de Massas , Células-Tronco Mesenquimais/imunologia , RNA Mensageiro/genética , Antígeno Sialil Lewis X
20.
Sci Rep ; 8(1): 420, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323143

RESUMO

While human Tregs hold immense promise for immunotherapy, their biologic variability poses challenges for clinical use. Here, we examined clinically-relevant activities of defined subsets of freshly-isolated and culture-expanded human PBMC-derived Tregs. Unlike highly suppressive but plastic memory Tregs (memTreg), naïve Tregs (nvTreg) exhibited the greatest proliferation, suppressive capacity after stimulation, and Treg lineage fidelity. Yet, unlike memTregs, nvTregs lack Fucosyltransferase VII and display low sLeX expression, with concomitant poor homing capacity. In vitro nvTreg expansion augmented their suppressive function, but did not alter the nvTreg sLeX-l°w glycome. However, exofucosylation of the nvTreg surface yielded high sLeX expression, promoting endothelial adhesion and enhanced inhibition of xenogeneic aGVHD. These data indicate that the immature Treg glycome is under unique regulation and that adult PBMCs can be an ideal source of autologous-derived therapeutic Tregs, provided that subset selection and glycan engineering are engaged to optimize both their immunomodulation and tropism for inflammatory sites.


Assuntos
Selectina E/metabolismo , Doença Enxerto-Hospedeiro/terapia , Leucócitos Mononucleares/citologia , Linfócitos T Reguladores/citologia , Animais , Proliferação de Células , Transplante de Células , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoterapia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Ligantes , Camundongos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante
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