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1.
Nanomaterials (Basel) ; 14(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38470778

RESUMO

High-resolution imaging of Cu/low-k on-chip interconnect stacks in advanced microelectronic products is demonstrated using full-field transmission X-ray microscopy (TXM). The comparison of two lens-based laboratory X-ray microscopes that are operated at two different photon energies, 8.0 keV and 9.2 keV, shows a contrast enhancement for imaging of copper nanostructures embedded in insulating organosilicate glass of a factor of 5 if 9.2 keV photons are used. Photons with this energy (Ga-Kα radiation) are generated from a Ga-containing target of a laboratory X-ray source applying the liquid-metal-jet technology. The 5 times higher contrast compared to the use of Cu-Kα radiation (8.0 keV photon energy) from a rotating anode X-ray source is caused by the fact that the energy of the Ga-Kα emission line is slightly higher than that of the Cu-K absorption edge (9.0 keV photon energy). The use of Ga-Kα radiation is of particular advantage for imaging of copper interconnects with dimensions from several 100 nm down to several 10 nm in a Cu/SiO2 or Cu/low-k backend-of-line stack. Physical failure analysis and reliability engineering in the semiconductor industry will benefit from high-contrast X-ray images of sub-µm copper structures in microchips.

2.
Nanomaterials (Basel) ; 14(2)2024 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-38276751

RESUMO

High-resolution imaging of buried metal interconnect structures in advanced microelectronic products with full-field X-ray microscopy is demonstrated in the hard X-ray regime, i.e., at photon energies > 10 keV. The combination of two multilayer optics-a side-by-side Montel (or nested Kirkpatrick-Baez) condenser optic and a high aspect-ratio multilayer Laue lens-results in an asymmetric optical path in the transmission X-ray microscope. This optics arrangement allows the imaging of 3D nanostructures in opaque objects at a photon energy of 24.2 keV (In-Kα X-ray line). Using a Siemens star test pattern with a minimal feature size of 150 nm, it was proven that features < 150 nm can be resolved. In-Kα radiation is generated from a Ga-In alloy target using a laboratory X-ray source that employs the liquid-metal-jet technology. Since the penetration depth of X-rays into the samples is significantly larger compared to 8 keV photons used in state-of-the-art laboratory X-ray microscopes (Cu-Kα radiation), 3D-nanopattered materials and structures can be imaged nondestructively in mm to cm thick samples. This means that destructive de-processing, thinning or cross-sectioning of the samples are not needed for the visualization of interconnect structures in microelectronic products manufactured using advanced packaging technologies. The application of laboratory transmission X-ray microscopy in the hard X-ray regime is demonstrated for Cu/Cu6Sn5/Cu microbump interconnects fabricated using solid-liquid interdiffusion (SLID) bonding.

3.
Nature ; 607(7920): 762-768, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35794484

RESUMO

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4+ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4+ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.


Assuntos
Linfócitos T CD4-Positivos , Alimentos , Tolerância Imunológica , Alérgenos/imunologia , Formação de Anticorpos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proteínas Alimentares/imunologia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/imunologia , Gliadina/imunologia , Tolerância Imunológica/imunologia , Inflamação , Interleucina-2/imunologia , Fígado/citologia , Fígado/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Fragmentos de Peptídeos/imunologia , Células T Auxiliares Foliculares/citologia , Células T Auxiliares Foliculares/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th1/citologia , Células Th1/imunologia
4.
JCI Insight ; 7(12)2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35730567

RESUMO

Immunosuppressed patients with inflammatory bowel disease (IBD) generate lower amounts of SARS-CoV-2 spike antibodies after mRNA vaccination than healthy controls. We assessed SARS-CoV-2 spike S1 receptor binding domain-specific (S1-RBD-specific) B lymphocytes to identify the underlying cellular defects. Patients with IBD produced fewer anti-S1-RBD antibody-secreting B cells than controls after the first mRNA vaccination and lower amounts of total and neutralizing antibodies after the second. S1-RBD-specific memory B cells were generated to the same degree in IBD and control groups and were numerically stable for 5 months. However, the memory B cells in patients with IBD had a lower S1-RBD-binding capacity than those in controls, which is indicative of a defect in antibody affinity maturation. Administration of a third shot to patients with IBD elevated serum antibodies and generated memory B cells with a normal antigen-binding capacity. These results show that patients with IBD have defects in the formation of antibody-secreting B cells and affinity-matured memory B cells that are corrected by a third vaccination.


Assuntos
COVID-19 , Doenças Inflamatórias Intestinais , Anticorpos Antivirais , COVID-19/prevenção & controle , Humanos , Células B de Memória , RNA Mensageiro , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
5.
Cell Rep ; 37(2): 109823, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34610291

RESUMO

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A+ subset and secondary MBCs that are mostly IgG+ and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses.


Assuntos
Vacinas contra COVID-19/imunologia , Plasmócitos/imunologia , SARS-CoV-2/imunologia , Adulto , Animais , Anticorpos Antivirais/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , COVID-19/metabolismo , Reações Cruzadas/imunologia , Feminino , Células HEK293 , Humanos , Imunização/métodos , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinação/métodos , Vacinas/imunologia
6.
PLoS Genet ; 17(8): e1009744, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424906

RESUMO

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality.


Assuntos
Proteínas de Drosophila/genética , Isolamento Reprodutivo , Animais , Centrômero/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila simulans/genética , Drosophila simulans/metabolismo , Genes Letais/genética , Especiação Genética , Hibridização Genética/genética , Reprodução/genética
7.
Nat Biotechnol ; 39(8): 943-948, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33941928

RESUMO

The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptide:MHC class I (p:MHCI) tetramers are well-established reagents for identifying antigen-specific CD8+ T cells by flow cytometry, but efforts to extend the approach to CD4+ T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that p:MHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4+ T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4+ T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.


Assuntos
Linfócitos T CD4-Positivos , Corantes Fluorescentes , Antígenos de Histocompatibilidade Classe II , Animais , Antígenos CD4/química , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
8.
Artigo em Inglês | MEDLINE | ID: mdl-33903156

RESUMO

Naive CD4+ T cells become memory cells after proliferating in response to their cognate major histocompatibility complex class II (MHCII)-bound peptide and passing through an effector cell stage. The process by which CD4+ memory T cells emerge from the effector cell pool, however, is less well understood than in the case of CD8+ T cells. During certain acute infections, naive CD4+ T cells proliferate and differentiate into various forms of type 1 (Th1) and follicular helper (Tfh) effector cells. We review the evidence that about 10% of the cells in each of these subsets survive to become memory cells that resemble their effector cell precursors. The roles that asymmetric cell division, the TCF-1 transcription factor, metabolic activity, reactive oxygen species, and the IL-7 receptor play in the effector to memory cell transition are discussed. We propose a speculative model in which the metabolic activity needed for rapid clonal expansion also generates toxic products that induce apoptosis in most effector cells. Memory cells then arise from the effector cells in each subset that are at the low end of the metabolic activity spectrum.


Assuntos
Linfócitos T CD8-Positivos , Memória Imunológica , Linfócitos T CD4-Positivos , Células T de Memória
9.
Immunity ; 54(4): 687-701.e4, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33773107

RESUMO

Interferon-γ (IFN-γ)-producing CD4+ T helper-1 (Th1) cells are critical for protection from microbes that infect the phagosomes of myeloid cells. Current understanding of Th1 cell differentiation is based largely on reductionist cell culture experiments. We assessed Th1 cell generation in vivo by studying antigen-specific CD4+ T cells during infection with the phagosomal pathogen Salmonella enterica (Se), or influenza A virus (IAV), for which CD4+ T cells are less important. Both microbes induced T follicular helper (Tfh) and interleukin-12 (IL-12)-independent Th1 cells. During Se infection, however, the Th1 cells subsequently outgrew the Tfh cells via an IL-12-dependent process and formed subsets with increased IFN-γ production, ZEB2-transcription factor-dependent cytotoxicity, and capacity to control Se infection. Our results indicate that many infections induce a module that generates Tfh and poorly differentiated Th1 cells, which is followed in phagosomal infections by an IL-12-dependent Th1 cell amplification module that is critical for pathogen control.


Assuntos
Diferenciação Celular/imunologia , Células Th1/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Drosophila/imunologia , Feminino , Interferon gama/imunologia , Interleucina-12/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/imunologia
10.
Complement Ther Med ; 45: 136-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31331550

RESUMO

OBJECTIVES: Infantile colic is a common benign disease occurring in early infancy that may have a great impact on family life. In the present study, the effectiveness and safety of the complex homeopathic medicine Enterokind was compared with Simethicone for treating infantile colic. DESIGN AND SETTING: Current data were drawn from a prospective, multicenter, randomized, open-label, controlled clinical trial that was conducted in 2009 in 3 Russian outpatient clinics. Children received either Enterokind (Chamomilla D6, Cina D6, Colocynthis D6, Lac defloratum D6 and Magnesium chloratum D6) or Simethicone. Data from infants ≤ 6 months with infantile colic are presented here. MAIN OUTCOME MEASURES: The main outcomes assessments were the change of total complaints score (maximum 17 points) and total objective symptoms score (maximum 22 points) after 10 days of treatment. RESULTS: Data from 125 infants ≤ 6 months with infantile colic were analyzed. The differences in total complaints and objective symptoms scores between baseline and day 10, estimated from the ANCOVA model, were found to be highly significant (p < 0.0001; ITT) in favor of Enterokind, both for complaints (Δ=-2.38; 95% confidence interval (CI): [-2.87; -1.89]) and for objective symptoms (Δ=-2.07; 95% CI: [-2.65; -1.49]). 1 adverse event (AE), vomiting, occurred under Enterokind and was rated to be unlikely related to it; 4 AEs occurred under Simethicone. All AEs were non-serious. CONCLUSIONS: The current study indicates that Enterokind is an effective and safe homeopathic treatment for functional intestinal colic in infants ≤ 6 months.


Assuntos
Cólica/tratamento farmacológico , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Homeopatia/métodos , Humanos , Lactente , Recém-Nascido , Estudos Prospectivos , Simeticone/uso terapêutico
11.
J Leukoc Biol ; 101(1): 329-338, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27493244

RESUMO

The liver contains 2 transcriptionally distinct group 1 ILC subsets: CD49a+ ILC1s and CD49b+ NK cells. However, little is known about how group 1 ILCs contribute to hepatic immune responses. Therefore, we characterized murine liver-resident group 1 ILCs and found that CD49a+ ILC1s express high levels of the inhibitory receptor NKG2A and localize near DCs in perivascular spaces surrounding the portal triads. Upon hepatic viral infection, NKG2A signaling in group 1 ILCs, especially in CD49a+ ILC1s, inhibits CXCL9 expression required for robust accumulation of IFN-γ+CD49b+ NK cells. As a consequence, NKG2A-/- mice showed increased numbers of IFN-γ-producing NK cells that preferentially activate liver CD103+ DCs, leading to the sustained proliferation of adoptively transferred, virus-specific CD8+ T cells. Collectively, these data suggest that group 1 ILCs play a role in maintaining the liver as a tolerogenic site by limiting the recruitment of peripheral NK cells during the early phase of viral infection. Furthermore, our findings implicate that the inhibition of NKG2A signaling on group 1 ILCs may be a novel vaccine strategy to induce robust CD8+ T cell responses against persistent liver pathogens.


Assuntos
Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Imunidade Inata , Fígado/citologia , Linfócitos/citologia , Adenoviridae/metabolismo , Animais , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Contagem de Células , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL9/biossíntese , Fatores Quimiotáticos/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Epitopos/imunologia , Feminino , Imunidade Inata/efeitos dos fármacos , Cadeias alfa de Integrinas/metabolismo , Integrina alfa1/metabolismo , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/deficiência , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo
12.
Hepatology ; 63(3): 799-812, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26662852

RESUMO

UNLABELLED: Nonalcoholic steatohepatitis (NASH) affects 3%-5% of the U.S. population, having severe clinical complications to the development of fibrosis and end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma. A critical cause of NASH is chronic systemic inflammation promoted by innate immune cells, such as liver macrophages (Mϕ) and natural killer (NK) cells. However, little is known about how the crosstalk between Mϕ and NK cells contributes to regulate NASH progression to fibrosis. In this report, we demonstrate that NKp46(+) cells play an important role in preventing NASH progression to fibrosis by regulating M1/M2 polarization of liver Mϕ. Using a murine model of NASH, we demonstrate that DX5(+)NKp46(+) NK cells are increased during disease and play a role in polarizing Mϕ toward M1-like phenotypes. This NK's immunoregulatory function depends on the production of interferon-gamma (IFN-γ), but not by granzyme-mediated cytolytic activity. Notably, depletion of NKp46(+) cells promotes the development of fibrosis with increased expression of profibrogenic genes as well as skewed M2 Mϕ phenotypes in hepatic tissues. CONCLUSIONS: NK cell-derived IFN-γ may be essential for maintaining a balanced inflammatory environment that promotes tissue integrity and limiting NASH progression to fibrosis.


Assuntos
Células Matadoras Naturais/fisiologia , Fígado/imunologia , Macrófagos/fisiologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Morte Celular , Colágeno/metabolismo , Progressão da Doença , Feminino , Fibrose , Células Estreladas do Fígado/metabolismo , Imunidade Inata , Interferon gama/metabolismo , Fígado/patologia , Ativação de Macrófagos , Camundongos Endogâmicos C57BL , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fenótipo , Receptor Cross-Talk , Linfócitos T Reguladores
13.
J Immunol ; 194(7): 3213-22, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25712214

RESUMO

The liver maintains a tolerogenic environment to avoid unwarranted activation of its resident immune cells upon continuous exposure to food and bacterially derived Ags. However, in response to hepatotropic viral infection, the liver's ability to switch from a hyporesponsive to a proinflammatory environment is mediated by select sentinels within the parenchyma. To determine the contribution of hepatic dendritic cells (DCs) in the activation of naive CD8(+) T cells, we first characterized resident DC subsets in the murine liver. Liver DCs exhibit unique properties, including the expression of CD8α (traditionally lymphoid tissue specific), CD11b, and CD103 markers. In both the steady-state and following viral infection, liver CD103(+) DCs express high levels of MHC class II, CD80, and CD86 and contribute to the high number of activated CD8(+) T cells. Importantly, viral infection in the Batf3(-/-) mouse, which lacks CD8α(+) and CD103(+) DCs in the liver, results in a 3-fold reduction in the proliferative response of Ag-specific CD8(+) T cells. Limiting DC migration out of the liver does not significantly alter CD8(+) T cell responsiveness, indicating that CD103(+) DCs initiate the induction of CD8(+) T cell responses in situ. Collectively, these data suggest that liver-resident CD103(+) DCs are highly immunogenic in response to hepatotropic viral infection and serve as a major APC to support the local CD8(+) T cell response. It also implies that CD103(+) DCs present a promising cellular target for vaccination strategies to resolve chronic liver infections.


Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Cadeias alfa de Integrinas/metabolismo , Fígado/imunologia , Ativação Linfocitária/imunologia , Adenoviridae/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Superfície/metabolismo , Antígeno CD11b/metabolismo , Movimento Celular , Feminino , Imunofenotipagem , Fígado/patologia , Fígado/virologia , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vírus/imunologia
14.
Hepatology ; 57(4): 1314-24, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23150092

RESUMO

UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Here we report that infection of hepatic cells by HCV stimulates nuclear factor kappa B (NFκB)-dependent production of thymic stromal lymphopoietin (TSLP). Hepatocyte-derived TSLP in turn conditions dendritic cells (DCs) to drive T-helper (Th)17 differentiation. The TSLP secreted by HCV-infected hepatoma cells is capable of activating human monocyte-derived DCs by up-regulating the expression of CD40, CD86, CCL17, CCL22, and CCL20 which are activating markers of DCs. In addition, the production of key cytokines for Th17 differentiation, transforming growth factor beta (TGF-ß), interleukin (IL)-6, and IL-21, is enhanced by human monocytes upon coculture with HCV-infected cells. Importantly, the blockade of TSLP using neutralizing antibody prevented the activation and maturation of DCs as well as the production of Th17 differentiation cytokines. DC conditioning by TSLP secreted from HCV-infected cells activated naïve CD4+ T lymphocytes, resulting in Th17 differentiation. Furthermore, we can detect substantial levels of hepatocyte TSLP in fibrotic liver tissue from chronic HCV patients. Thus, blockade of TSLP released by HCV-infected hepatocytes may suppress the induction/maintenance of hepatic Th17 responses and halt the progression of chronic liver disease to fibrosis and liver failure. CONCLUSION: Hepatocyte-derived TSLP conditions DCs to drive Th17 differentiation. Treatment of TSLP neutralizing antibody in HCV-infected hepatocyte/DC coculture abrogates DC conditioning and thereby inhibits Th17 differentiation.


Assuntos
Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Citocinas/metabolismo , Hepacivirus/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Células Th17/patologia , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Comunicação Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Citocinas/antagonistas & inibidores , Citocinas/efeitos dos fármacos , Células Dendríticas/patologia , Hepatite C/patologia , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Monócitos/patologia , NF-kappa B/metabolismo , Células Th17/metabolismo , Linfopoietina do Estroma do Timo
15.
Blood ; 118(24): 6332-41, 2011 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-21908428

RESUMO

Fc receptor-like 4 (FcRL4) is expressed on the surface of a subset of memory B cells (MBCs) located at sites of invading pathogens in mucosal lymphoid tissues in healthy individuals. Recently, FcRL4(+) MBCs were shown to be greatly increased in number in the peripheral blood of HIV-infected viremic individuals, in whom they are associated with B-cell exhaustion, and in individuals chronically reinfected with malaria. In the present study, we provide evidence that the expression of FcRL4 in human B-cell lines disrupts immune synapse formation and blocks antigen-induced BCR signaling at the point of Syk phosphorylation, blocking downstream activation of PLC-γ2 and Vav and the induction of calcium responses and CD69 expression. FcRL4 functions by ligation-independent mechanisms that require the 3 tyrosine residues in its cytoplasmic domain and involves its phosphorylation and association with the tyrosine phosphatases SHP-1 and SHP-2. Remarkably, FcRL4 is concentrated in endosomes after treatment with the TLR9 agonist CpG and enhances signaling through TLR9, as measured by increased expression of CD23. These findings suggest that FcRL4 may act as a molecular switch in B cells to dampen adaptive immune signaling and enhance innate signaling in response to chronic antigenic stimulation.


Assuntos
Linfócitos B/metabolismo , Regulação para Baixo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Receptores Fc/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Regulação para Cima , Linfócitos B/citologia , Linfócitos B/imunologia , Linhagem Celular , Humanos , Sinapses Imunológicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Ativação Linfocitária , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores Fc/química , Receptores Fc/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Quinase Syk , Receptor Toll-Like 9/antagonistas & inibidores
16.
Crit Rev Immunol ; 31(1): 43-52, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21395510

RESUMO

NK cells represent a large proportion of the lymphocyte population in the liver and are involved in early innate immunity to pathogen infection. As a result of liver endothelial cell fenestrations, parenchymal cells are not separated by a basal membrane, and thereby pathogen-infected hepatocytes are extensively capable of interacting with innate immune cells including NK cells. In addition, hepatic NK cells interact with surrounding DC and alter their differentiation and function. Recent studies reveal that NK cells exhibit a regulatory function that modulates T cell responses through their interaction with DC and/or direct effect on T cells. Thus, NK cells play a central role, not only in innate immunity, but also in shaping the adaptive immune response. During pathogen infection, there is a remarkable increase of hepatic NK cells, possibly due to the expansion of resident liver NK cells and/or recruitement of NK cells from the blood. The liver microenvironment is believed to modulate hepatic NK cell function through the induction of activating/inhibitory receptor expression and inflammatory cytokine secretion. Particularly, the liver maintains intrahepatic NK cells in a functionally hyporesponsive state compared to splenic NK cells: liver NK cells displayed a dampened IFN-γ response to IL-12/IL-18 stimulation. Notably, the liver contains a significant population of functionally hyporesponsive NK cells that express high levels of the inhibitory receptor NKG2A and lack expression of MHC class I-binding Ly49 receptors. Importantly, adoptively transferred splenic NK cells that migrate to the liver displayed phenotypic and functional changes, supporting a view that the liver environment modifies NK cell receptor expression and functional responsiveness. In this article, we will review studies on the regulation of NK cell repertoire and function in the hepatic environment and the impact of liver NK cell immunoregulatory function on influencing adaptive immunity.


Assuntos
Células Matadoras Naturais/imunologia , Fígado/imunologia , Imunidade Adaptativa , Animais , Diferenciação Celular , Humanos , Células Matadoras Naturais/citologia , Receptores de Células Matadoras Naturais/imunologia
17.
Structure ; 18(8): 934-43, 2010 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-20696394

RESUMO

The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igbeta form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igalpha displays a similar fold as Igbeta. An Igalphabeta heterodimer model was generated based on the unique disulfide-bonded Igbeta dimer. Solution binding studies showed that the extracellular domains of Igalphabeta preferentially recognize the constant region of BCR with mu chain specificity, suggesting a role for Igalphabeta to enhance BCRmu chain signaling. Cluster mutations on Igalpha, Igbeta, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cmu4 domain of mIgM. These studies provide the first structural model for understanding BCR function.


Assuntos
Antígenos CD79/química , Modelos Moleculares , Conformação Proteica , Receptores de Antígenos de Linfócitos B/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD79/metabolismo , Cristalografia , Análise Mutacional de DNA , Primers do DNA/genética , Dimerização , Humanos , Camundongos , Dados de Sequência Molecular , Dobramento de Proteína , Receptores de Antígenos de Linfócitos B/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Ressonância de Plasmônio de Superfície
18.
Immunity ; 30(1): 44-55, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19135393

RESUMO

B cells are activated in vivo after the B cell receptors (BCRs) bind to antigens captured on the surfaces of antigen-presenting cells. Antigen binding results in BCR microclustering and signaling; however, the molecular nature of the signaling-active BCR clusters is not well understood. Using single-molecule imaging techniques, we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 domains alone clustered spontaneously and activated B cells. These results support a unique mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR, revealing an interface that promotes BCR clustering.


Assuntos
Linfócitos B/imunologia , Membrana Celular/fisiologia , Regiões Constantes de Imunoglobulina/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Nitro-Hidroxi-Iodofenilacetato/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase Syk
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