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1.
Nat Commun ; 12(1): 5074, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417463

RESUMO

ß cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human ß cells with inflammation but its expression is reduced in surviving ß cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate ß cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO ß cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between ß cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in ß cells may reduce activating pathologic immune cells and killing of ß cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 1/patologia , Inflamação/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Citotoxicidade Imunológica , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Feminino , Humanos , Imunidade , Inflamação/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Transcrição Genética
2.
Curr Opin Immunol ; 69: 29-38, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33640598

RESUMO

Immune checkpoint inhibitors have revolutionized the treatments of cancers but are also associated with immune related adverse events that can interfere with their use. The types and severity of adverse events vary with checkpoint inhibitors. A single mechanism of pathogenesis has not emerged: postulated mechanisms involve direct effects of the checkpoint inhibitor, emergence of autoantibodies or autoreactive T cells, and destruction by toxic effects of activated T cells. Several host factors such as genotypes, preexisting autoimmune disease, inflammatory responses and others may have predictive value. Ongoing investigations seek to identify ways of modulating the autoimmunity without affecting the anti-tumor response with agents that are specific for the autoimmune mechanisms.

3.
Nat Biotechnol ; 39(5): 619-629, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33558698

RESUMO

Current methods for comparing single-cell RNA sequencing datasets collected in multiple conditions focus on discrete regions of the transcriptional state space, such as clusters of cells. Here we quantify the effects of perturbations at the single-cell level using a continuous measure of the effect of a perturbation across the transcriptomic space. We describe this space as a manifold and develop a relative likelihood estimate of observing each cell in each of the experimental conditions using graph signal processing. This likelihood estimate can be used to identify cell populations specifically affected by a perturbation. We also develop vertex frequency clustering to extract populations of affected cells at the level of granularity that matches the perturbation response. The accuracy of our algorithm at identifying clusters of cells that are enriched or depleted in each condition is, on average, 57% higher than the next-best-performing algorithm tested. Gene signatures derived from these clusters are more accurate than those of six alternative algorithms in ground truth comparisons.


Assuntos
Biologia Computacional , Análise de Sequência de RNA/tendências , Análise de Célula Única/tendências , Transcriptoma/genética , Algoritmos , Análise por Conglomerados , Simulação por Computador , Humanos , Funções Verossimilhança
4.
Annu Rev Med ; 72: 313-330, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-32886542

RESUMO

Immune checkpoint inhibitors (CPIs) reverse immune suppression that is thought to allow malignant growth. Despite remarkable efficacy in a subset of cancers, their use is accompanied by immune-related adverse events, including endocrinopathies such as hypophysitis, thyroid dysfunction, diabetes, and adrenalitis. These conditions are heterogenous, with differing incidence across CPI types, but are unified by the acuity and extremity of tissue-specific organ failure. Their occurrence may be associated with beneficial tumor control. Further understanding of the risk factors and mechanisms of these endocrine immunotoxicities can help optimize CPI use as well as improve understanding of spontaneous autoimmune diseases.

5.
Mol Metab ; 27S: S104-S113, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500821

RESUMO

BACKGROUND: The extended and clinically silent progression of Type 1 diabetes (T1D) creates a challenge for clinical interventions and for understanding the mechanisms that underlie its pathogenesis. Over the course of the development of Type 1 diabetes, studies in animal models and of human tissues have identified adaptive changes in ß cells that may affect their immunogenicity and susceptibility to killing. Loss of ß cells has traditionally been identified by impairment in function but environmental factors may affect these measurements. SCOPE OF REVIEW: In this review we will highlight features of ß cell responses to cell death, particularly in the setting of inflammation, and focus on methods of detecting ß cell death in vivo. MAJOR CONCLUSIONS: We developed an assay to measure ß cell death in vivo by detecting cell free DNA with epigenetic modifications of the INS gene that are found in ß cells. This assay has robust technical performance and identifies killing in individuals at very high risk for disease, but its ability to identify ß cell killing in at-risk relatives is limited by the short half-life of the cell free DNA and the need for repeated sampling over an extended course. We present results from the Diabetes Prevention Trial-1 using this assay. In addition, recent studies have identified cellular adaptations in some ß cells that may avoid killing but impair metabolic function. Cells with these characteristics may aggravate the autoimmune response but also may represent a potentially recoverable source of functional ß cells.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Humanos
7.
Diabetologia ; 62(4): 655-664, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30569273

RESUMO

AIMS/HYPOTHESIS: The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of 'partially exhausted' CD8+ T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years. METHODS: Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release. RESULTS: Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8+ T cells at follow-up. CONCLUSIONS/INTERPRETATION: These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab. TRIAL REGISTRATION: ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Adolescente , Adulto , Área Sob a Curva , Autoimunidade , Peptídeo C/sangue , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Criança , Citocinas/metabolismo , Feminino , Seguimentos , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Masculino , Ensaios Clínicos Controlados Aleatórios como Assunto , Indução de Remissão , Resultado do Tratamento , Adulto Jovem
8.
J Immunol ; 201(6): 1662-1670, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30082321

RESUMO

Type 1 diabetes (T1D) is most likely caused by killing of ß cells by autoreactive CD8+ T cells. Methods to isolate and identify these cells are limited by their low frequency in the peripheral blood. We analyzed CD8+ T cells, reactive with diabetes Ags, with T cell libraries and further characterized their phenotype by CyTOF using class I MHC tetramers. In the libraries, the frequency of islet Ag-specific CD45RO+IFN-γ+CD8+ T cells was higher in patients with T1D compared with healthy control subjects. Ag-specific cells from the libraries of patients with T1D were reactive with ZnT8186-194, whereas those from healthy control recognized ZnT8186-194 and other Ags. ZnT8186-194-reactive CD8+ cells expressed an activation phenotype in T1D patients. We found TCR sequences that were used in multiple library wells from patients with T1D, but these sequences were private and not shared between individuals. These sequences could identify the Ag-specific T cells on a repeated draw, ex vivo in the IFN-γ+ CD8+ T cell subset. We conclude that CD8+ T cell libraries can identify Ag-specific T cells in patients with T1D. The T cell clonotypes can be tracked in vivo with identification of the TCR gene sequences.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Células Secretoras de Insulina/patologia , Masculino
9.
Diabetes ; 67(8): 1471-1480, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29937434

RESUMO

Insulin-dependent diabetes may occur in patients with cancers who are treated with checkpoint inhibitors (CPIs). We reviewed cases occurring over a 6-year period at two academic institutions and identified 27 patients in whom this developed, or an incidence of 0.9%. The patients had a variety of solid-organ cancers, but all had received either anti-PD-1 or anti-PD-L1 antibodies. Diabetes presented with ketoacidosis in 59%, and 42% had evidence of pancreatitis in the peridiagnosis period. Forty percent had at least one positive autoantibody and 21% had two or more. There was a predominance of HLA-DR4, which was present in 76% of patients. Other immune adverse events were seen in 70%, and endocrine adverse events in 44%. We conclude that autoimmune, insulin-dependent diabetes occurs in close to 1% of patients treated with anti-PD-1 or -PD-L1 CPIs. This syndrome has similarities and differences compared with classic type 1 diabetes. The dominance of HLA-DR4 suggests an opportunity to identify those at highest risk of these complications and to discover insights into the mechanisms of this adverse event.


Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Doenças Autoimunes/induzido quimicamente , Antígeno B7-H1/antagonistas & inibidores , Diabetes Mellitus Tipo 1/etiologia , Modelos Imunológicos , Pancreatite/induzido quimicamente , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/uso terapêutico , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/fisiopatologia , Antígeno B7-H1/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Genótipo , Antígeno HLA-DR4/sangue , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Insulina/uso terapêutico , Secreção de Insulina , Isoanticorpos/análise , Cetose/etiologia , Cetose/prevenção & controle , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Pâncreas/metabolismo , Pancreatite/imunologia , Pancreatite/metabolismo , Pancreatite/fisiopatologia , Receptor de Morte Celular Programada 1/metabolismo
10.
Curr Opin Immunol ; 49: 44-50, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28992525

RESUMO

Studies with immunologics have shown that the natural history of Type 1 diabetes can be modified. These studies have targeted key mediators of the disease and recent analyses, together with studies in preclinical models have identified mechanisms that may be involved in the clinical effects. Several issues remain including specificity of the interventions, adverse effects of the treatments, and duration of their effects. Future studies are likely to include more specific approaches with agents such as cell therapies with selected immune regulatory subsets, antigen specific therapies, and combinations of agents with complementary mechanisms of activity.


Assuntos
Autoantígenos/uso terapêutico , Produtos Biológicos/uso terapêutico , Diabetes Mellitus Tipo 1/terapia , Imunoterapia/métodos , Células Secretoras de Insulina/patologia , Animais , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Células Secretoras de Insulina/efeitos dos fármacos , Terapia de Alvo Molecular
11.
Cell Metab ; 25(3): 727-738, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28190773

RESUMO

Type 1 diabetes (T1D) is a chronic autoimmune disease that involves immune-mediated destruction of ß cells. How ß cells respond to immune attack is unknown. We identified a population of ß cells during the progression of T1D in non-obese diabetic (NOD) mice that survives immune attack. This population develops from normal ß cells confronted with islet infiltrates. Pathways involving cell movement, growth and proliferation, immune responses, and cell death and survival are activated in these cells. There is reduced expression of ß cell identity genes and diabetes antigens and increased immune inhibitory markers and stemness genes. This new subpopulation is resistant to killing when diabetes is precipitated with cyclophosphamide. Human ß cells show similar changes when cultured with immune cells. These changes may account for the chronicity of the disease and the long-term survival of ß cells in some patients.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Animais , Anticorpos Monoclonais/farmacologia , Complexo CD3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Feminino , Humanos , Imunoterapia , Células Secretoras de Insulina/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos Endogâmicos NOD , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
12.
Front Immunol ; 6: 654, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26834735

RESUMO

Regulatory T cells (Tregs) control unwanted immune responses, including those that mediate tolerance to self as well as to foreign antigens. Their mechanisms of action include direct and indirect effects on effector T cells and important functions in tissue repair and homeostasis. Tregs express a number of cell surface markers and transcriptional factors that have been instrumental in defining their origins and potentially their function. A number of immune therapies, such as rapamycin, IL-2, and anti-T cell antibodies, are able to induce Tregs and are being tested for their efficacy in diverse clinical settings with exciting preliminary results. However, a balance exists with the use of some, such as IL-2, that may have effects on unwanted populations as well as promoting expansion and survival of Tregs requiring careful selection of dose for clinical use. The use of cell surface markers has enabled investigators to isolate and expand ex vivo Tregs more than 500-fold routinely. Clinical trials have begun, administering these expanded Tregs to patients as a means of suppressing autoimmune and alloimmune responses and potentially inducing immune tolerance. Studies in the future are likely to build on these initial technical achievements and use combinations of agents to improve the survival and functional capacity of Tregs.

13.
Mol Cell Neurosci ; 46(1): 235-44, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20869442

RESUMO

Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG's inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or overexpression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.


Assuntos
Córtex Cerebral/citologia , Glicoproteína Associada a Mielina/metabolismo , Neuritos/fisiologia , Neurônios/citologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Células CHO , Células Cultivadas , Técnicas de Cocultura , Cricetinae , Cricetulus , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Associada a Mielina/genética , Neuritos/ultraestrutura , Neurônios/fisiologia , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho
14.
J Virol ; 81(2): 532-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16943294

RESUMO

The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.


Assuntos
Éteres/farmacologia , Produtos do Gene env/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Hidrocarbonetos Fluorados/farmacologia , Fusão de Membrana/efeitos dos fármacos , Piperazinas/farmacologia , Animais , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Enfuvirtida , Proteína gp41 do Envelope de HIV/farmacologia , Humanos , Peso Molecular , Fragmentos de Peptídeos/farmacologia
15.
J Biol Chem ; 280(23): 22172-80, 2005 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-15817447

RESUMO

Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.


Assuntos
Endotélio Vascular/citologia , Regulação da Expressão Gênica , Microcirculação/metabolismo , Trombina/química , Trombospondina 1/biossíntese , Difosfato de Adenosina/química , Algoritmos , Amidas/farmacologia , Apoptose , Células Cultivadas , Análise por Conglomerados , Meios de Cultura , Primers do DNA/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Impedância Elétrica , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Modelos Biológicos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/química , Toxina Pertussis/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Piridinas/farmacologia , RNA/metabolismo , Receptor PAR-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombina/metabolismo , Fatores de Tempo , Veias Umbilicais/citologia , Regulação para Cima
16.
J Virol ; 78(7): 3742-52, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15016894

RESUMO

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the "phenylalanine 43 cavity" and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Piperazinas/farmacologia , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Cães , Farmacorresistência Viral/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/fisiologia , Humanos , Modelos Moleculares , Fenilalanina/genética , Fenilalanina/metabolismo , Estrutura Terciária de Proteína
17.
J Virol ; 78(4): 1873-81, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14747552

RESUMO

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.


Assuntos
Aminoácido Oxirredutases/metabolismo , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/patogenicidade , Hepatite Viral Animal/virologia , Animais , Carboxipeptidases/metabolismo , Células Cultivadas , Patos , Glicina Desidrogenase (Descarboxilante) , Hepatócitos/enzimologia , Hepatócitos/virologia , Virulência
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