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1.
PLoS Pathog ; 17(5): e1009576, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015061

RESUMO

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.


Assuntos
COVID-19/transmissão , Lectinas Tipo C/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Antígenos CD/metabolismo , COVID-19/prevenção & controle , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Chlorocebus aethiops , Humanos , Células Jurkat , Pulmão/metabolismo , Lectinas de Ligação a Manose/metabolismo , Manosídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Mucosa Respiratória/metabolismo , Células Vero
2.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805444

RESUMO

Macrophage colony-stimulating factor (M-CSF or CSF-1) is known to have a broad range of actions on myeloid cells maturation, including the regulation of macrophage differentiation, proliferation and survival. Macrophages generated by M-CSF stimulus have been proposed to be alternatively activated or M2 phenotype. M-CSF is commonly overexpressed by tumors and is also known to enhance tumor growth and aggressiveness via stimulating pro-tumor activities of tumor-associated macrophages (TAMs). Currently, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. However, its antitumor activity shows a limited single-agent effect. Therefore, macrophages in response to M-CSF interruption are pending for further investigation. To achieve this study, bone marrow derived macrophages were generated in vitro by M-CSF stimulation for 7 days and then continuously grown until day 21 in M-CSF absence. A selective pressure for cell survival was initiated after withdrawal of M-CSF. The surviving cells were more prone to M2-like phenotype, even after receiving interleukin-4 (IL-4) stimulation. The transcriptome analysis unveiled that endogenous CSF-1 level was dramatically up-regulated and numerous genes downstream to CSF-1 covering tumor necrosis factor (TNF), ras-related protein 1 (Rap1) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway were significantly modulated, especially for proliferation, migration and adhesion. Moreover, the phenomenal increase of miR-21-5p and genes related to pro-tumor activity were observed in parallel. In summary, withholding of CSF-1/CSF-1R interaction would rather augment than suspend the M-CSF-driven pro-tumor activities of M2 macrophages in a long run.


Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/patologia
3.
Nat Commun ; 12(1): 2147, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846309

RESUMO

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).


Assuntos
Canal Anal/citologia , Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Genitália/citologia , HIV-1/fisiologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Monócitos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Forma Celular , Colagenases/metabolismo , Derme/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Membrana Mucosa/metabolismo , Fagócitos/metabolismo , Fenótipo , Receptores CCR5/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transcrição Genética
4.
Eur J Pharmacol ; 901: 174097, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848540

RESUMO

Renal fibrosis is the common pathological hallmark of chronic kidney disease, and SET domain containing lysine methyltransferase 7 (SETD7) promote considerably renal fibrosis. However, the signaling mechanisms underlying SETD7 driving renal fibrosis are not fully understood. Here, we investigated the role of SETD7 in M2 macrophages-myofibroblasts transition and the myeloid fibroblasts activation in folic acid and obstruction-induced renal fibrosis. Mice treated with PFI-2, an inhibitor of SETD7, presented less bone marrow-derived myofibroblasts, fewer CD206+/α-smooth muscle actin + cells and developed less renal fibrosis (P<0.01). Furthermore, SETD7 inhibition reduced the infiltration of inflammatory cells and decreased the production of pro-inflammatory cytokines and chemokines in the kidneys after folic acid treatment (P<0.01). Finally, SETD7 inhibition suppressed the accumulation of NF-κB p65+ cells in folic acid nephropathy (P<0.01). Taken together, SETD7 mediates M2 macrophages-myofibroblasts transition, bone marrow-derived myofibroblasts activation, and inflammation response in the development of renal fibrosis.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Ácido Fólico/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Nefropatias/tratamento farmacológico , Rim/patologia , Animais , Fibroblastos/efeitos dos fármacos , Fibrose , Nefropatias/induzido quimicamente , Nefropatias/patologia , Testes de Função Renal , Lectinas Tipo C/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Fator de Transcrição RelA/efeitos dos fármacos
5.
Acta Trop ; 217: 105870, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33636152

RESUMO

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Antígenos de Bactérias/metabolismo , Linfócitos B/imunologia , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismo , Simulação de Acoplamento Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologia , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Vacinas contra a Tuberculose/metabolismo , Vacinas de Subunidades/química , Vacinas de Subunidades/imunologia
6.
PLoS Negl Trop Dis ; 14(9): e0008626, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898175

RESUMO

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.


Assuntos
Células Dendríticas/metabolismo , Vesículas Extracelulares/metabolismo , Fasciola hepatica/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Fenótipo , Animais , Antígenos de Helmintos/análise , Biomarcadores , Medula Óssea , Citocinas/metabolismo , Modelos Animais de Doenças , Fasciola hepatica/isolamento & purificação , Fasciolíase/imunologia , Fasciolíase/parasitologia , Glicoproteínas , Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismo , Proteômica , Linfócitos T/imunologia
7.
Mol Immunol ; 126: 129-135, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32823237

RESUMO

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.


Assuntos
Desenvolvimento Embrionário/imunologia , Proteínas de Peixes/metabolismo , Imunidade Materno-Adquirida , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Takifugu/crescimento & desenvolvimento , Animais , Embrião não Mamífero , Feminino , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Lactococcus/imunologia , Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Óvulo/imunologia , Óvulo/metabolismo , Receptores de Superfície Celular/imunologia , Streptococcus iniae/imunologia , Takifugu/imunologia , Takifugu/microbiologia
8.
Nat Commun ; 11(1): 4064, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792542

RESUMO

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2LMW-/- mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS+/CD206+ TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Radioterapia/métodos , Animais , Linhagem Celular Tumoral , Fator 2 de Crescimento de Fibroblastos/genética , Células HT29 , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/efeitos da radiação , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Life Sci ; 258: 118085, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663578

RESUMO

BACKGROUND: An integral intestinal barrier is essential for intestinal homeostasis. Yet, as a side effect of cancer treatment, chemotherapeutic drugs have been reported to cause mucositis. In a recent study, we found that alginate oligosaccharides (AOS) prevent busulfan induced intestinal mucositis. However, it is not known if AOS improves small intestine epithelial cell integrity and migration, which are two essential processes for maintaining the mechanical barrier function of the small intestine. In the current investigation, we aimed to explore the effects of AOS on the integrity and migration of small intestine cells using swine intestinal epithelial IPEC-J2 cells. METHODS: Cell integrity was determined using the TEER assay. Cell migration capability was detected using a wound healing experiment. Small interfering RNA (siRNA) was used to inhibit mannose receptor (MR) expression. Western blotting and immunofluorescence staining were used to determine protein expression. RESULTS: Increasing levels of AOS improved cell integrity as measure by TEER. At the same time, AOS improved IPEC-J2 cell migration capacity as shown in the wound closure assay. It is interesting to note that AOS increased the expression of intestinal microvillus proteins and junction proteins to benefit cell integrity. MR siRNA blocked the action of AOS on cell integrity and cell migration and inhibited the expression of microvillus and cell junction proteins. CONCLUSION: We identified the underlying mechanisms by which AOS improved small intestinal mucositis. As a novel, natural food additive, AOS may be administered to prevent intestinal mucositis induced by chemotherapy or other issues.


Assuntos
Alginatos/farmacologia , Movimento Celular/efeitos dos fármacos , Intestino Delgado/citologia , Oligossacarídeos/farmacologia , Animais , Linhagem Celular , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Miosinas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/metabolismo , Suínos , Proteínas de Junções Íntimas/metabolismo , Cicatrização/efeitos dos fármacos
10.
Proc Natl Acad Sci U S A ; 117(23): 12980-12990, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461368

RESUMO

The aryl hydrocarbon receptor (AhR) represents an environmental sensor regulating immune responses. In the skin, AhR is expressed in several cell types, including keratinocytes, epidermal Langerhans cells (LC), and dermal dendritic cells (DC). The mechanisms how AhR activates or inhibits cutaneous immune responses remain controversial, owing to differences in the cell-specific functions of AhR and the different activating ligands. Therefore, we sought to investigate the role of AhR in LC and langerin+ and negative DC in the skin. To this aim, we generated Langerin-specific and CD11c-specific knockout (-/-) mice lacking AhR, respectively, in LC and Langerin+ dermal DC and in all CD11c+ cells. These were then tested in an epicutaneous protein (ovalbumin, Ova) sensitization model. Immunofluorescence microscopy and flow cytometry revealed that Langerin-AhR-/- but not CD11c-AhR-/- mice harbored a decreased number of LC with fewer and stunted dendrites in the epidermis as well as a decreased number of LC in skin-draining lymph nodes (LN). Moreover, in the absence of AhR, we detected an enhanced T helper type-2 (Th2) [increased interleukin 5 (IL-5) and interleukin 13 (IL-13)] and T regulatory type-1 (Tr1) (IL-10) response when LN cells were challenged with Ova in vitro, though the number of regulatory T cells (Treg) in the LN remained comparable. Langerin-AhR-/- mice also exhibited increased blood levels of Ova-specific immunoglobulin E (IgE). In conclusion, deletion of AhR in langerin-expressing cells diminishes the number and activation of LC, while enhancing Th2 and Tr1 responses upon epicutaneous protein sensitization.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células de Langerhans/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Administração Cutânea , Animais , Antígenos de Superfície/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Epiderme/imunologia , Epiderme/metabolismo , Técnicas de Inativação de Genes , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Células de Langerhans/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Receptores de Hidrocarboneto Arílico/genética , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismo
11.
Am J Respir Crit Care Med ; 201(10): 1209-1217, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32197050

RESUMO

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.


Assuntos
Fumar Cigarros/patologia , Pulmão/patologia , Macrófagos Alveolares/patologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Células , Feminino , Humanos , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Leucossialina/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Dispositivos Ópticos , Receptores de Superfície Celular/metabolismo , Doadores de Tecidos
12.
BMC Cancer ; 20(1): 192, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32143591

RESUMO

BACKGROUND: Altered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the glycosylation. METHODS: We analyzed resected HCC specimens (n = 50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type N-glycans. RESULTS: Four lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor prognosis. CONCLUSION: Dedifferentiation of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Concanavalina A/metabolismo , Neoplasias Hepáticas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Lectinas de Plantas/metabolismo , Idoso , Calystegia/química , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Intervalo Livre de Doença , Feminino , Glicosilação , Humanos , Imuno-Histoquímica/métodos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , N-Acetilglucosaminiltransferases/metabolismo , Narcissus/química , Imagem Óptica/métodos , Polissacarídeos/química , Coloração e Rotulagem/métodos
13.
Nat Commun ; 11(1): 1368, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170195

RESUMO

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.


Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/fisiologia , Eritropoetina/metabolismo , Fator V , Fator VIII/metabolismo , Glicoproteínas/genética , Complexo de Golgi/fisiologia , Humanos , Lectinas de Ligação a Manose/metabolismo , Transporte Proteico , Via Secretória
14.
Sci Rep ; 10(1): 2642, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060374

RESUMO

Mesenchymal stromal cells (MSC) have immunomodulatory effects impacting macrophages, promoting polarisation towards a reparative phenotype. CCL2 is a potent cytokine involved in the recruitment of macrophages. We hypothesised that MSC derived CCL2 may be involved in the MSC therapeutic effect by facilitating macrophage repolarisation. To further delineate this mechanism, MSC isolated from CCL2 deficient mice (MSC-KO) were applied to excisional wounds in wild-type (WT) mice. CCL2 deficiency in MSC completely abrogated the therapeutic response compared to MSC-WT. MSC-KO were unable to repolarise macrophages to the same extent as WT and this was accompanied by a reduced angiogenesis and re-epithelialisation of the wounds at day 10. This study demonstrates that MSC derived CCL2 is required for MSC induced accelerated wound healing. The role of CCL2 in the interaction between MSC and Macrophages has not been previously demonstrated in accelerated wound healing. CCL2 has a potent effect on the ability to reduce the inflammatory response through local recruitment of macrophages. This research highlights CCL2 as a possible target for augmentation of MSC therapy to enhance therapeutic potential.


Assuntos
Quimiocina CCL2/metabolismo , Células-Tronco Mesenquimais/metabolismo , Cicatrização , Animais , Polaridade Celular , Modelos Animais de Doenças , Feminino , Imunidade Inata , Imunomodulação , Inflamação/patologia , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Fisiológica , Reepitelização , Receptores de Superfície Celular/metabolismo
15.
Immunol Cell Biol ; 98(2): 152-164, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31845380

RESUMO

Depending on the microenvironment conditions, macrophages display phenotypic and functional heterogeneity. This study characterized the programmed cell death-ligand 2 (PD-L2)-expressing macrophage-like cells drained from surgical wound zones, and investigated their influence on helper T (Th) cell responses. Although all CD14+ myeloid cells possessed macrophage-like features, CD206+ and CD163+ cells constituted a specific subpopulation with high PD-L2 expression. There was a modest correlation between the PD-L2 levels on CD206+ macrophages and the amount of interferon (IFN)-γ in the drainage fluid. The adhesion-independent macrophages simultaneously presented both classically-activated M1 and alternatively-activated M2 characteristics. CD206+ and PD-L2+ cells were identified with high granularity and size, expressed arginase-1 and costimulatory molecules, had enhanced phagocytic activity and produced reactive oxygen species. The genes associated with macrophage differentiation (MERTK, AXL and TYRO3) were also upregulated. These cells provided costimulation to Th cells; yet, when PD-L2 was blocked, T-cell proliferation and IFNγ production were enhanced. Under defined conditions devoid of activation stimuli and matrix adhesion, ex vivo-generated monocyte-derived macrophages displayed limited capacity to stimulate T cells. Upon exposure to IFNγ, they significantly upregulated programmed death 1 ligands, especially PD-L2. These cells did not completely abrogate T-cell differentiation; however, PD-L2 checkpoint blockade restored Th1 proliferation and secretion of interleukin-2, tumor necrosis factor-α and IFNγ. In conclusion, upregulation of PD-L2 on the wound zone macrophages may constitute a negative feedback loop that restrains the Th1 effector responses and avoids exacerbation of inflammation during tissue healing.


Assuntos
Diferenciação Celular/imunologia , Inflamação/metabolismo , Macrófagos/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Células Th1/imunologia , Cicatrização/fisiologia , Adulto , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Arginase/metabolismo , Antígeno B7-H1/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Interferon gama/farmacologia , Interleucina-2/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th1/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização/efeitos dos fármacos , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo
16.
Diabetes ; 69(3): 401-412, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31843955

RESUMO

M2 macrophages play an important role in tissue repair and regeneration. They have also been found to modulate ß-cell replication in mouse models of pancreatic injury and disease. We previously reported that ß-cell replication is strongly increased in a subgroup of human organ donors characterized by prolonged duration of stay in an intensive care unit (ICU) and increased number of leukocytes in the pancreatic tissue. In the present study we investigated the relationship between duration of stay in the ICU, M2 macrophages, vascularization, and pancreatic cell replication. Pancreatic organs from 50 donors without diabetes with different durations of stay in the ICU were analyzed by immunostaining and digital image analysis. The number of CD68+CD206+ M2 macrophages increased three- to sixfold from ≥6 days' duration of stay in the ICU onwards. This was accompanied by a threefold increased vascular density and a four- to ninefold increase in pancreatic cells positive for the replication marker Ki67. A strong correlation was observed between the number of M2 macrophages and ß-cell replication. These results show that a prolonged duration of stay in the ICU is associated with an increased M2 macrophage number, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data show evidence of marked levels of tissue repair in the human donor pancreas.


Assuntos
Proliferação de Células/fisiologia , Unidades de Terapia Intensiva , Tempo de Internação , Macrófagos/patologia , Pâncreas/fisiologia , Regeneração/fisiologia , Doadores de Tecidos , Adolescente , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , Pâncreas/metabolismo , Pâncreas/patologia , Receptores de Superfície Celular/metabolismo , Adulto Jovem
17.
Macromol Rapid Commun ; 41(1): e1900459, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31721357

RESUMO

The synthesis of brush glycopolymers mimicking the architecture of proteoglycans is achieved by grafting sequence-defined glycooligomers derived from solid-phase polymer synthesis onto a poly(active ester) scaffold. This approach gives access to a first library of brush glycopolymers with controlled variations in the degree of branching and number of carbohydrate ligands per branch. When studying lectin binding of linear and brush glycopolymers to lectins Concanavalin A (ConA), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and mannose-binding lectin (MBL), different preferences are observed with MBL showing higher binding to linear glycopolymer and ConA and DC-SIGN favoring brush glycopolymers. This finding suggests that the architecture of polymeric glycan mimetics affects binding to lectins not only in terms of creating higher avidity but potentially also selectivity ligands.


Assuntos
Moléculas de Adesão Celular/metabolismo , Concanavalina A/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Catálise , Moléculas de Adesão Celular/química , Concanavalina A/química , Cobre/química , Lectinas Tipo C/química , Lectinas de Ligação a Manose/química , Polímeros/síntese química , Polímeros/química , Ligação Proteica , Receptores de Superfície Celular/química , Ressonância de Plasmônio de Superfície
18.
Carbohydr Polym ; 229: 115404, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826490

RESUMO

Biomaterial-host interactions significantly affect tissue repair, which is modulated by macrophages. In this study, a polysaccharide, konjac glucomannan (KGM), was acetylated with different degrees of substitution (DS), and the acetylated KGM (AceKGM)-based fibrous membrane was designed to modulate the activity of macrophages for accelerating wound healing. AceKGM was biocompatible and easily dissolved in organic solvents. The adhesion force between Raw264.7 cells and the AceKGM substrate was quantitatively detected by atomic force microscopy (AFM). The enzyme-linked immunosorbent assay (ELISA) results showed that the AceKGM fibrous membrane enhanced macrophage expression of anti-inflammatory and pro-regenerative cytokines, and the DS of AceKGM significantly affected membrane bioactivity. The full-thickness mouse skin wound repair experiments indicated that the AceKGM-containing fibrous membranes significantly accelerated wound healing by promoting re-epithelialization, tissue remodeling, and collagen deposition. In summary, AceKGM-based fibrous membranes have potential as bioactive scaffolds for wound regeneration.


Assuntos
Bandagens , Mananas , Membranas Artificiais , Cicatrização , Acetilação , Animais , Adesão Celular , Lectinas Tipo C/metabolismo , Mananas/química , Lectinas de Ligação a Manose/metabolismo , Camundongos , Células NIH 3T3 , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo
19.
Immunology ; 159(1): 63-74, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573680

RESUMO

Monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte-derived macrophages were generated by 5-day culture with macrophage and granulocyte-macrophage colony-stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1-skewed (CD80high  HLA-DRhigh  CD163low ), whereas negative selection MDMs were M2-skewed (CD80low  HLA-DRlow  CD163high ). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.


Assuntos
Diferenciação Celular , Separação Celular/métodos , Citometria de Fluxo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/fisiologia , Monócitos/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores , Adesão Celular , Células Cultivadas , Humanos , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
J Asthma ; 57(1): 1-10, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30588853

RESUMO

Objective: Local cytokine milieu (especially Th2 inflammatory type) secreted into the asthmatic airways affect the alternative activated macrophages polarization (M2). TSLP and IL-33 are important alarmins of allergic response associated with Th2 inflammation. The aim of the study was to investigate the expression of the receptors for epithelial derived cytokines: TSLP (TSLPR) and IL-33 (ST2) on induced sputum CD206 positive macrophages from asthma and healthy subjects and analyze the relationships between these receptors and clinical features of the disease. Methods: Immunofluorescence staining for CD206 and TSLPR or ST2 on sputum macrophages was performed in 20 adult patients with stable asthma - 75% with atopy (3 intermittent, 12 mild-to-moderate, 5 severe, of which 11 were on biological anty-IgE treatment) and 23 healthy adult controls - 48% with atopy. Results: Our study demonstrated an increased expression of TSLP and IL-33 receptors on bronchial CD206 positive macrophages in asthma group. TSLPR but not ST2 had also greater expression on CD206 negative macrophages in asthma patients. Increased expression of both investigated receptors was related to longer disease duration and impaired lung function. We observed increased count of CD206lowTSLPhigh macrophages as well as positive correlation of these cells with total serum IgE in patients with atopy. Conclusions: The macrophage response during allergic reaction is likely to be connected with TSLP but rather not with IL-33 action. Our study indicates an important role of crosstalk between macrophages, TSLP and IL-33 in asthma pathophysiology.


Assuntos
Asma/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Macrófagos/imunologia , Receptores de Citocinas/metabolismo , Adulto , Asma/patologia , Estudos Transversais , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Interleucina-33/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Superfície Celular/metabolismo , Receptores de Citocinas/imunologia , Escarro/citologia , Escarro/imunologia
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