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1.
Sci Immunol ; 6(61)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326184

RESUMO

The spillover of animal coronaviruses (aCoVs) to humans has caused SARS, MERS, and COVID-19. While antibody responses displaying cross-reactivity between SARS-CoV-2 and seasonal/common cold human coronaviruses (hCoVs) have been reported, potential cross-reactivity with aCoVs and the diagnostic implications are incompletely understood. Here, we probed for antibody binding against all seven hCoVs and 49 aCoVs represented as 12,924 peptides within a phage-displayed antigen library. Antibody repertoires of 269 recovered COVID-19 patients showed distinct changes compared to 260 unexposed pre-pandemic controls, not limited to binding of SARS-CoV-2 antigens but including binding to antigens from hCoVs and aCoVs with shared motifs to SARS-CoV-2. We isolated broadly reactive monoclonal antibodies from recovered COVID-19 patients that bind a shared motif of SARS-CoV-2, hCoV-OC43, hCoV-HKU1, and several aCoVs, demonstrating that interspecies cross-reactivity can be mediated by a single immunoglobulin. Employing antibody binding data against the entire CoV antigen library allowed accurate discrimination of recovered COVID-19 patients from unexposed individuals by machine learning. Leaving out SARS-CoV-2 antigens and relying solely on antibody binding to other hCoVs and aCoVs achieved equally accurate detection of SARS-CoV-2 infection. The ability to detect SARS-CoV-2 infection without knowledge of its unique antigens solely from cross-reactive antibody responses against other hCoVs and aCoVs suggests a potential diagnostic strategy for the early stage of future pandemics. Creating regularly updated antigen libraries representing the animal coronavirome can provide the basis for a serological assay already poised to identify infected individuals following a future zoonotic transmission event.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Biblioteca de Peptídeos , Adolescente , Adulto , Idoso , Animais , Infecções por Coronavirus/diagnóstico , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Zoonoses
2.
Sci Immunol ; 6(60)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088746

RESUMO

In this issue of Science Immunology, Gallman et al. reveal how S-geranylgeranyl-l-glutathione cleavage and transport support P2RY8-driven B cell confinement to the germinal centers and its role in lymphocyte homing to the bone marrow.

3.
Nat Chem Biol ; 17(9): 954-963, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33972797

RESUMO

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
5.
J Exp Med ; 217(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31873727

RESUMO

Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites.


Assuntos
Centro Germinativo/metabolismo , Plasmócitos/metabolismo , Quinase Syk/metabolismo , Animais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proliferação de Células/fisiologia , Expressão Gênica/fisiologia , Centro Germinativo/fisiologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia
6.
J Exp Med ; 216(11): 2515-2530, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31492809

RESUMO

Germinal centers (GCs) are sites wherein B cells proliferate and mutate their immunoglobulins in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). Here, we mapped the location of single B cells in the context of intact lymph nodes (LNs) throughout the GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we demonstrated that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response.


Assuntos
Linfócitos B/metabolismo , Proliferação de Células , Centro Germinativo/metabolismo , Linfonodos/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Linfócitos B/citologia , Movimento Celular , Centro Germinativo/citologia , Linfonodos/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência
7.
Nat Immunol ; 20(4): 482-492, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833793

RESUMO

Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Seleção Clonal Mediada por Antígeno , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologia
8.
Science ; 358(6370): 1622-1626, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29217582

RESUMO

Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways.


Assuntos
Linfócitos B/imunologia , Perfilação da Expressão Gênica/métodos , Células Matadoras Naturais/imunologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Genes Reporter/efeitos dos fármacos , Genômica/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Neoplasias/imunologia , Baço/imunologia , Baço/virologia , Viroses/imunologia
9.
J Exp Med ; 214(11): 3435-3448, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-28939548

RESUMO

The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T-B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh-B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.


Assuntos
Antígenos CD/imunologia , Linfócitos B/imunologia , Moléculas de Adesão Celular/imunologia , Comunicação Celular/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Antígenos CD/genética , Antígenos CD/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Clonais/imunologia , Células Clonais/metabolismo , Citometria de Fluxo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo
10.
Cell Rep ; 18(3): 685-699, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28099847

RESUMO

The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM). Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers in vitro and in vivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not. We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlaced between endothelial stress fibers in order to complete TEM.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Leucócitos/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Amidas/farmacologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Interleucina-1beta/farmacologia , Leucócitos/citologia , Contração Muscular/efeitos dos fármacos , Miosina Tipo II/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Piridinas/farmacologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Imagem com Lapso de Tempo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
11.
FASEB J ; 30(5): 1767-78, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26823454

RESUMO

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8(+) and CD4(+) effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils.-Petrovich, E., Feigelson, S. W., Stoler-Barak, L., Hatzav, M., Solomon, A., Bar-Shai, A., Ilan, N., Li, J.-P., Engelhardt, B., Vlodavsky, I., Alon, R. Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Inflamação/induzido quimicamente , Molécula 1 de Adesão Intercelular/metabolismo , Pneumopatias/induzido quimicamente , Linfócitos/fisiologia , Neutrófilos/fisiologia , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Movimento Celular , Endotoxinas/toxicidade , Regulação da Expressão Gênica/fisiologia , Glucuronidase/metabolismo , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão Intercelular/genética , Pulmão/irrigação sanguínea , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos
12.
FASEB J ; 29(5): 2010-21, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25634957

RESUMO

Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that TH1/TC1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent. Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum.


Assuntos
Endotélio Vascular/imunologia , Glucuronidase/fisiologia , Inflamação/imunologia , Neutrófilos/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Feminino , Citometria de Fluxo , Inflamação/enzimologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/enzimologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/enzimologia , Pele/patologia , Linfócitos T/citologia , Linfócitos T/enzimologia
13.
PLoS One ; 9(1): e85699, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465652

RESUMO

A hallmark of immune cell trafficking is directional guidance via gradients of soluble or surface bound chemokines. Vascular endothelial cells produce, transport and deposit either their own chemokines or chemokines produced by the underlying stroma. Endothelial heparan sulfate (HS) was suggested to be a critical scaffold for these chemokine pools, but it is unclear how steep chemokine gradients are sustained between the lumenal and ablumenal aspects of blood vessels. Addressing this question by semi-quantitative immunostaining of HS moieties around blood vessels with a pan anti-HS IgM mAb, we found a striking HS enrichment in the basal lamina of resting and inflamed post capillary skin venules, as well as in high endothelial venules (HEVs) of lymph nodes. Staining of skin vessels with a glycocalyx probe further suggested that their lumenal glycocalyx contains much lower HS density than their basolateral extracellular matrix (ECM). This polarized HS pattern was observed also in isolated resting and inflamed microvascular dermal cells. Notably, progressive skin inflammation resulted in massive ECM deposition and in further HS enrichment around skin post capillary venules and their associated pericytes. Inflammation-dependent HS enrichment was not compromised in mice deficient in the main HS degrading enzyme, heparanase. Our results suggest that the blood vasculature patterns steep gradients of HS scaffolds between their lumenal and basolateral endothelial aspects, and that inflammatory processes can further enrich the HS content nearby inflamed vessels. We propose that chemokine gradients between the lumenal and ablumenal sides of vessels could be favored by these sharp HS scaffold gradients.


Assuntos
Membrana Basal/metabolismo , Vasos Sanguíneos/metabolismo , Endotélio Vascular/metabolismo , Heparitina Sulfato/metabolismo , Animais , Células CHO , Capilares/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Cricetinae , Cricetulus , Dermatite/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Matriz Extracelular/metabolismo , Feminino , Glucuronidase/deficiência , Glucuronidase/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Pele/irrigação sanguínea , Pele/patologia , Vênulas/metabolismo
14.
Int Immunol ; 26(6): 315-24, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24402310

RESUMO

Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1ß stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.


Assuntos
Endotélio Vascular/metabolismo , Heparina/análogos & derivados , N-Acetilglucosaminiltransferases/metabolismo , Neutrófilos/imunologia , Proteoglicanas/metabolismo , Linfócitos T/imunologia , Linhagem Celular , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiotaxia , Heparina/metabolismo , Humanos , Inflamação/imunologia , Interleucina-1beta/metabolismo , N-Acetilglucosaminiltransferases/genética , RNA Interferente Pequeno/genética , Migração Transendotelial e Transepitelial/genética , Fator de Necrose Tumoral alfa
15.
Innate Immun ; 20(7): 697-711, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24107515

RESUMO

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.


Assuntos
Doenças da Medula Óssea/genética , Doenças da Medula Óssea/terapia , Endotoxinas/toxicidade , Transplante de Células-Tronco Hematopoéticas , Polimorfismo de Nucleotídeo Único/genética , Proteínas de Fase Aguda/genética , Proteínas de Transporte/genética , Quimiocinas/metabolismo , Estudos de Coortes , Genótipo , Humanos , Receptores de Lipopolissacarídeos/genética , Glicoproteínas de Membrana/genética , Fator de Necrose Tumoral alfa/metabolismo
16.
Pediatr Res ; 72(2): 203-11, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22580716

RESUMO

BACKGROUND: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates. METHODS: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay. RESULTS: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1ß protein. Expression of S100A12 protein was localized to TA neutrophils. CONCLUSION: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population.


Assuntos
Endotoxinas/imunologia , Imunidade Inata/imunologia , Recém-Nascido/imunologia , Respiração Artificial/efeitos adversos , Proteínas S100/metabolismo , Traqueia/metabolismo , Fatores Etários , Análise de Variância , Citocinas/metabolismo , Endotoxinas/metabolismo , Citometria de Fluxo , Humanos , Teste do Limulus , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Proteína S100A12 , Traqueia/microbiologia
17.
J Allergy Clin Immunol ; 130(1): 195-204.e9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22521247

RESUMO

BACKGROUND: Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported. OBJECTIVE: We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs. METHODS: Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA and Western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist-induced cytokine responses was defined in rhesus macaque whole blood ex vivo. RESULTS: IMQs were more potent and effective than alum at inducing TNF and IL-1ß from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1ß production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 IMQs induced robust TNF and IL-1ß in whole blood of rhesus macaques at birth and infancy. CONCLUSIONS: IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust monocyte and MoDC activation and represent promising neonatal adjuvants.


Assuntos
Adenosina/metabolismo , Caspase 1/metabolismo , Células Dendríticas/imunologia , Imidazóis/farmacologia , Monócitos/imunologia , Quinolinas/farmacologia , Receptor 8 Toll-Like/agonistas , Adjuvantes Imunológicos , Adulto , Compostos de Alúmen , Animais , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Recém-Nascido , Macaca mulatta
18.
Nat Immunol ; 13(1): 67-76, 2011 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-22138716

RESUMO

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.


Assuntos
Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Linfócitos/imunologia , Migração Transendotelial e Transepitelial/imunologia , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Integrinas/metabolismo , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Camundongos , Receptores CCR2/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/imunologia , Vasculite/metabolismo
19.
Sci Transl Med ; 3(110): 110ra118, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22116933

RESUMO

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.


Assuntos
Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Proteínas Sanguíneas/uso terapêutico , Medula Óssea/patologia , Fluoroquinolonas/uso terapêutico , Lesões por Radiação/tratamento farmacológico , Técnicas de Ablação , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/farmacologia , Contagem de Células Sanguíneas , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Citocinas/sangue , Endotoxemia/sangue , Endotoxemia/complicações , Endotoxinas/metabolismo , Enrofloxacina , Fluoroquinolonas/administração & dosagem , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Transplante de Células-Tronco Hematopoéticas , Humanos , Mediadores da Inflamação/sangue , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutropenia/sangue , Neutropenia/complicações , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/efeitos da radiação , Lesões por Radiação/sangue , Lesões por Radiação/complicações , Análise de Sobrevida , Irradiação Corporal Total
20.
PLoS One ; 5(4): e10111, 2010 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-20404927

RESUMO

BACKGROUND: Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24-48 h) were markedly impaired in TLR2-deficient mice. CONCLUSIONS/SIGNIFICANCE: TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.


Assuntos
Bacteriemia/imunologia , Staphylococcus epidermidis/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Anticorpos , Sangue/microbiologia , Células Cultivadas , Citocinas/biossíntese , Imunidade Inata , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Infecções Estafilocócicas/imunologia , Receptor 2 Toll-Like/deficiência
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