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CD63 e CD123 expressão, autoanticorpos IgG e acurácia do teste do soro autólogo em pacientes com urticária crônica

Calamita, Zamir; Antunes, Roseli Nunes da Silveira; Almeida Filho, Odilon Marques de; Baleotti Júnior, Wilson; Calamita, Andrea Bronhara Pelá; Fukasawa, Josianne Thomazini; Cavaretto, Débora de Aguiar
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 21-28
Português
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Introduction: The autologous serum skin test (ASST) may suggest an autoimmune etiology in chronic urticaria (CU). A new laboratory technique called basophil activation test (BAT) has been currently employed for its diagnosis. Objective: To analyze ASST in relation to BAT as well as to evaluate interleukin 3 (IL3) receptors (CD123) and non-specific immunoglobulin G (IgG) autoantibodies bound to basophils in patients with chronic urticaria. Methods: We studied 33 adults with CU and mean age of 42.5 + 14 years. After stimulation by serum from patients with CU, CD63 expression on basophils from one atopic donor was analyzed by flow cytometry. Furthermore, we investigated CD123 and IgG autoantibody expressions. Results: The odds ratio (OR) between ASST and BAT was 1.00 (95% confidence interval [CI]: 0.22 to 4.5). The ASST for autoimmune CU diagnosis showed an accuracy of 54.5%, sensitivity of 66%, specificity of 33%, positive predictive value of 63%, and negative predictive value of 36%. There was no statistical difference between the studied groups as to mean non-specific IgG and CD123 expressions (for a p < 0.05). Discussion: This study demonstrated that ASST has low accuracy in the diagnosis of autoimmune CU. Concerning other analyzed aspects...

CD63 e CD123 expressão, autoanticorpos IgG e acurácia do teste do soro autólogo em pacientes com urticária crônica

Calamita,Zamir; Antunes,Roseli Nunes da Silveira; Almeida Filho,Odilon Marques de; Baleotti Júnior,Wilson; Calamita,Andrea Bronhara Pelá; Fukasawa,Josianne Thomazini; Cavaretto,Débora de Aguiar
Fonte: Sociedade Brasileira de Patologia Clínica; Sociedade Brasileira de Patologia; Sociedade Brasileira de Citopatologia Publicador: Sociedade Brasileira de Patologia Clínica; Sociedade Brasileira de Patologia; Sociedade Brasileira de Citopatologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/2012 Português
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INTRODUÇÃO: Na urticária crônica (UC), o teste cutâneo do soro autólogo (TCSA) pode sugerir a etiologia autoimune. Recentemente, uma nova técnica laboratorial denominada teste de ativação de basófilos (TAB) vem sendo utilizada para esse diagnóstico. OBJETIVOS: Analisar o TCSA em relação ao TAB, assim como avaliar os receptores da interleucina 3 (IL3) (CD123) e a presença de autoanticorpos da classe de imunoglobulina G (IgG) inespecíficos ligados aos basófilos de pacientes com UC. MÉTODOS: Estudamos 33 adultos com UC espontânea com idade média de 42,5 + 14 anos. Por meio da citometria de fluxo foi feita a análise da expressão das moléculas CD63 em basófilos de um doador atópico após o estímulo pelo soro dos pacientes com UC. Também realizamos a pesquisa da expressão da molécula CD123 e de autoanticorpos IgG inespecíficos. RESULTADOS: O odds ratio (OR) entre o TCSA e o TAB foi de 1 (intervalo de confiança [IC] 95%: 0,22-4,5). O TCSA para o diagnóstico da UC autoimune mostrou acurácia de 54,5%, sensibilidade de 66%, especificidade de 33%, valor preditivo positivo de 63% e valor preditivo negativo de 36%. Não houve diferença estatística entre os grupos estudados quanto à média de expressão dos anticorpos IgG inespecíficos e das moléculas CD123 (para um p < 0...

The Tetraspanin CD63/lamp3 Cycles between Endocytic and Secretory Compartments in Human Endothelial Cells

Kobayashi, Toshihide; Vischer, Ulrich M.; Rosnoblet, Corinne; Lebrand, Cécile; Lindsay, Margaret; Parton, Robert G.; Kruithof, Egbert K. O.; Gruenberg, Jean
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /05/2000 Português
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In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular–multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel–Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel–Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel–Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel–Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel–Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel–Palade bodies.

Role of Adaptor Complex AP-3 in Targeting Wild-Type and Mutated CD63 to Lysosomes

Rous, Brian A.; Reaves, Barbara J.; Ihrke, Gudrun; Briggs, John A.G.; Gray, Sally R.; Stephens, David J.; Banting, George; Luzio, J. Paul
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /03/2002 Português
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CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit μ3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with μ3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells. Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with μ3 but not at all with μ2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells. In contrast, it localized to the cell surface in transfected cells of pearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the δ subunit of AP-3. Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes. Using this AP-3–dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from the trans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes.

The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.

Nishibori, M; Cham, B; McNicol, A; Shalev, A; Jain, N; Gerrard, J M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1993 Português
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The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491...

The tetraspanin CD63 enhances the internalization of the H,K-ATPase β-subunit

Duffield, Amy; Kamsteeg, Erik-Jan; Brown, Andrea N.; Pagel, Philipp; Caplan, Michael J.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase β-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The β-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the β-subunit from the cell surface to CD63+ intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase β-subunit complexed with CD63. Coexpression of the β-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the β-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.

Syntenin-1 Is a New Component of Tetraspanin-Enriched Microdomains: Mechanisms and Consequences of the Interaction of Syntenin-1 with CD63▿

Latysheva, Nadya; Muratov, Gairat; Rajesh, Sundaresan; Padgett, Matthew; Hotchin, Neil A.; Overduin, Michael; Berditchevski, Fedor
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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Tetraspanins are clustered in specific microdomains (named tetraspanin-enriched microdomains, or TERM) in the plasma membrane and regulate the functions of associated transmembrane receptors, including integrins and receptor tyrosine kinases. We have identified syntenin-1, a PDZ domain-containing protein, as a new component of TERM and show that syntenin-1 specifically interacts with the tetraspanin CD63. Detailed biochemical and heteronuclear magnetic resonance spectroscopy (NMR) studies have demonstrated that the interaction is mediated by the C-terminal cytoplasmic region of the tetraspanin and the PDZ domains of syntenin-1. Upon interaction, NMR chemical shift perturbations were predominantly localized to residues around the binding pocket of PDZ1, indicating a specific mode of recognition of the cytoplasmic tail of CD63. In addition, the C terminus of syntenin-1 has a stabilizing role in the CD63-syntenin-1 association, as deletion of the last 17 amino acids abolished the interaction. The CD63-syntenin-1 complex is abundant on the plasma membrane, and the elevated expression of the wild-type syntenin-1 slows down constitutive internalization of the tetraspanin. Furthermore, internalization of CD63 was completely blocked in cells expressing a syntenin-1 mutant lacking the first 100 amino acids. Previous results have shown that CD63 is internalized via AP-2-dependent mechanisms. Hence...

Diclofenac induces basophil degranulation without increasing CD63 expression in sensitive patients

Malbrán, A; Yeyati, E; Rey, G L; Galassi, N
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /01/2007 Português
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Diclofenac (Dc) induces an IgE-independent basophil (Ba) degranulation in susceptible individuals. CD63 Ba expression is utilized as an in vitro test for diagnosis of drug hypersensitivity. We tested the ability of Dc to induce CD63 Ba expression by flow cytometry (BAT) and Ba degranulation using light microscopy (HBDT) in patients sensitive to Dc. We studied 14 patients with diclofenac hypersensitivity, also two patients sensitive to Dermatophagoides pteronyssinus (Dp), and 12 normal controls. HBDT was performed by mononuclear cells toluidine blue staining. BAT determined CD63 expression in antiCD63/anti-IgE/anti-CD45-labelled whole blood. In each case, the percentage of activated Ba post-stimulation with 1 and 10 µg/ml Dc was determined. Positive controls included N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide-induced activation. IgE-mediated Ba activation was induced with a Dp allergenic extract. With Dc 1 µg/ml, mean HBDT in Dc-susceptible individuals was 33·62 ± 18·35% and 8·49 ± 4·79% in controls (P = 0·0001). Mean BAT was 2·04 ± 1·68% and 1·93 ± 1·40% in controls (P = 0·8). Ba preincubation with Dc did not affect fMLP-induced CD63 expression, neither in Dc-sensitive individuals (P = 0·8) (n = 4) nor in subjects without Dc hypersensitivity (P = 0·25) (n = 4). Ba from the two patients sensitive both to Dc and Dp responded to Dp but not to Dc by BAT: Dc...

CD63 Interacts with the Carboxy-Terminus of the Colonic H+,K+-ATPase to Increase Plasma Membrane Localization and Rb+-Uptake

Codina, Juan; Li, Jian; DuBose, Thomas D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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The carboxy-terminus of the colonic H+,K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the carboxy-terminus of the α-subunit of mouse colonic H+,K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize interaction of CT-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro, incubated, and complexes immunoprecipitated. CT-HKα 2 protein (but not CT-HKα1) co-precipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+,K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1 and 86Rb+-uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2/NKβ1 complex in the cell membrane.

CD63 Is Not Required for Production of Infectious Human Immunodeficiency Virus Type 1 in Human Macrophages▿

Ruiz-Mateos, Ezequiel; Pelchen-Matthews, Annegret; Deneka, Magdalena; Marsh, Mark
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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During the assembly of human immunodeficiency virus type 1 (HIV-1) particles, the tetraspanin CD63 can be incorporated into the viral membrane. Indeed, cell surface tetraspanin microdomains that include CD63 have been proposed as sites for virus release. In addition, antibodies against CD63 can inhibit HIV infection of macrophages. In this cell type, HIV assembles into intracellularly sequestered plasma membrane domains that contain several other tetraspanins, including CD81, CD9, and CD53. CD63 is recruited to this domain following HIV infection. Together, these observations suggest that CD63 may have some function in the assembly of infectious virus particles and/or the infectivity of assembled virions. Here we have used RNA interference to knock down CD63 expression in monocyte-derived primary macrophages. We show that in the absence of CD63, HIV assembly is quantitatively comparable to that seen in CD63-expressing macrophages and that virus assembly occurs on compartments positive for CD81, CD9, and CD53. Moreover, the infectivity of macrophage-derived virus is unaffected by the loss of CD63. Together, our results indicate that at least in tissue culture, CD63 expression is not required for either the production or the infectivity of HIV-1.

Deficiency of the Tetraspanin CD63 Associated with Kidney Pathology but Normal Lysosomal Function▿

Schröder, Jenny; Lüllmann-Rauch, Renate; Himmerkus, Nina; Pleines, Irina; Nieswandt, Bernhard; Orinska, Zane; Koch-Nolte, Friedrich; Schröder, Bernd; Bleich, Markus; Saftig, Paul
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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CD63 is a member of the tetraspanin superfamily that constitutes a main component of the lysosomal membrane. In mice, two CD63 gene loci are present, with only one of these two being functional. We generated and analyzed mice deficient for active CD63. Disruption of CD63 results in a complete loss of CD63 protein expression. Despite its abundance in late endosomes/lysosomes, the lack of CD63 does not cause obvious endosomal/lysosomal abnormalities. CD63 knockout mice are viable and fertile without gross morphological abnormalities in the majority of tissues. No alterations in the populations of immune cells and only minor differences in platelet function were observed. This suggests that the lack of CD63 could be successfully compensated for, most likely by other tetraspanins. However, CD63 deficiency leads to an altered water balance. CD63 knockout mice show an increased urinary flow, water intake, reduced urine osmolality, and a higher fecal water content. In principle cells of the collecting duct of CD63-deficient mice, abnormal intracellular lamellar inclusions were observed. This indicates that the sorting of apical transport proteins might be impaired in these cells. CD63 knockout mice provide an important tool for analyzing the various postulated functions of CD63 in vivo.

A Critical Role for CD63 in HIV Replication and Infection of Macrophages and Cell Lines

Chen, Hui; Dziuba, Natallia; Friedrich, Brian; von Lindern, Jana; Murray, James L.; Rojo, Daniel R.; Hodge, Thomas W.; O’Brien, William A.; Ferguson, Monique R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.

Ameloblastin Regulates Osteogenic Differentiation by Inhibiting Src Kinase via Cross Talk between Integrin β1 and CD63 ▿

Iizuka, Shinji; Kudo, Yasusei; Yoshida, Maki; Tsunematsu, Takaaki; Yoshiko, Yuji; Uchida, Takashi; Ogawa, Ikuko; Miyauchi, Mutsumi; Takata, Takashi
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Ameloblastin, the most abundant nonamelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here, we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin were abrogated by treatment with anti-CD63 antibody and overexpression of constitutively active Src, respectively. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1.

A Post-Entry Role for CD63 in Early HIV-1 Replication

Li, Guangyu; Dziuba, Natallia; Friedrich, Brian; Murray, James L.; Ferguson, Monique R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Macrophages and CD4+ lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4+ cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.

Marked Differences in the Signaling Requirements for Expression of CD203c and CD11b versus CD63 Expression and Histamine Release in Human Basophils

MacGlashan, Donald
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Many techniques are being used to examine the status of circulating human basophils including the enhanced expression of a variety of cell surface proteins. There is accumulating evidence that there are at least two compartments containing these activation marker proteins but there are only some indications for the signaling requirements for each of the compartments. The current studies began with published reports by other investigators that potentially dissociated CD63 expression from anaphylactic degranulation with the p38 inhibitor, SB203580, a possible falsification of a previously proposed hypothesis regarding CD63 expression. The current studies examined regulation of activation marker expression to explore signaling requirements. First, it was found that inhibition of both histamine release and CD63 expression with SB203580 was concordant. But it was also found that this agent had no effect on increased expression of CD203c and CD11b. Actin polymerization inhibitors caused marked enhancement of CD63 expression (concordant with their effects on degranulation) with no effect on expression of CD203c and CD11b. The third generation syk inhibitor, NVP-QAB205, showed 5-fold lower potency for inhibiting expression of CD203c and CD11b than CD63. Finally...

The tetraspanin CD63 is required for efficient IgE-mediated mast cell degranulation and anaphylaxis1

Kraft, Stefan; Jouvin, Marie-Hélène; Kulkarni, Nitin; Kissing, Sandra; Morgan, Ellen S.; Dvorak, Ann M.; Schröder, Bernd; Saftig, Paul; Kinet, Jean-Pierre
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Mast cell activation through the high affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. While antibodies against the tetraspanins CD63 and CD81 inhibit FcεRI-induced mast cell degranulation, the intrinsic role of these molecules in FcεRI-induced mast cell activation is unknown. In mast cells, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). Here, we investigated the role of CD63 in mast cells using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo mast cell numbers and tissue distribution. Bone-marrow-derived mast cells (BMMC) developed normally in the absence of CD63 protein. However, CD63-deficient BMMC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by β-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon mast cell activation, was also decreased. IL-6 secretion, and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression...

Tetraspanin CD63 is a regulator of HIV-1 replication

Fu, Enqing; Pan, Lei; Xie, Yonghong; Mu, Deguang; Liu, Wei; Jin, Faguang; Bai, Xuefan
Fonte: e-Century Publishing Corporation Publicador: e-Century Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em 01/02/2015 Português
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Macrophages and CD4+ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4+ T-cells, dendritic cells, and a CD4+ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4+ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4+ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2).

Identifizierung und Charakterisierung neuer Aktivierungsmarker auf basophilen Granulozyten; Identification and characterization of novel activation markers on basophilic granulocytes

Hennersdorf, Immanuel Florian
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Basophile und Mastzellen sind wichtige Effektorzellen entzündlicher Reaktionen. Im Unterschied zu Eosinophilen und Neutrophilen verfügen sie über hochaffine Immunglobulin (Ig) E-Rezeptoren, die durch Bindung von Allergenen an rezeptorgebundenes IgE vernetzt und dadurch aktiviert werden. Dies führt zur Ausschüttung verschiedener Mediatoren in den Extrazellulärraum. Zusätzlich kommt es zur Verschiebung von in Granula gespeicherten Membranproteinen wie CD63 und dem Ektoenzym CD203c an die Plasmamembran. Diese Aktivierungsmarker werden inzwischen routinemäßig in durchflußzytometrischen Basophilenaktivierungstests angewendet. Die Größe der durch die beiden Marker CD63 und CD203c erkannten Populationen unterscheidet sich deutlich. So zeigt nur eine Subpopulation der Zellen die auf Allergenstimulation CD203c hochregulieren auch eine gesteigerte Oberflächenexpression von CD63. Außerdem unterscheidet sich das kinetische Profil der Hochregulierung von CD63 sowie die Sensitivität gegenüber Inhibitoren und Aktivatoren der durch FcRI vermittelten Signaltransduktion gegenüber CD203c. Kürzlich wurde gezeigt, dass Tetradecanoylphorbolacetat (TPA), ein Aktivator der Proteinkinase C (PKC), zu einer beschleunigten und verstärkten Hochregulierung von CD203c...

Determination of protein regions responsible for interactions of amelogenin with CD63 and LAMP1

Zou, YanMing; Wang, HongJun; Shapiro, Jason L.; Okamoto, Curtis T.; Brookes, Steven J.; Lyngstadaas, S. Petter; Snead, Malcolm L.; Paine, Michael L.
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
Português
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The enamel matrix protein amelogenin is secreted by ameloblasts into the extracellular space to guide the formation of highly ordered hydroxyapatite mineral crystallites, and, subsequently, is almost completely removed during mineral maturation. Amelogenin interacts with the transmembrane proteins CD63 and LAMP (lysosome-associated membrane protein) 1, which are involved in endocytosis. Exogenously added amelogenin has been observed to move rapidly into CD63/LAMP1-positive vesicles in cultured cells. In the present study, we demonstrate the protein region defined by amino acid residues 103–205 for CD63 interacts not only with amelogenin, but also with other enamel matrix proteins (ameloblastin and enamelin). A detailed characterization of binding regions in amelogenin, CD63 and LAMP1 reveals that the amelogenin region defined by residues PLSPILPELPLEAW is responsible for the interaction with CD63 through residues 165–205, with LAMP1 through residues 226–251, and with the related LAMP2 protein through residues 227–259. We predict that the amelogenin binding region is: (i) hydrophobic; (ii) largely disordered; and (iii) accessible to the external environment. In contrast, the binding region of CD63 is likely to be organized in a ‘7’ shape within the mushroom-like structure of CD63 EC2 (extracellular domain 2). In vivo...

Avaliação da expressão dos marcadores CD63 e CD203c em basófilos e do seu contributo no diagnóstico de hipersensibilidade ao diclofenac

Moscoso,Teresa; Melo,Alcinda Campos; Neto,Marta; Santos,Maria Conceição Pereira
Fonte: Sociedade Portuguesa de Alergologia e Imunologia Clínica Publicador: Sociedade Portuguesa de Alergologia e Imunologia Clínica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2014 Português
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37.59%
Introdução: O grupo dos anti‑inflamatorios não esteróides (AINEs) é um dos grupos farmacológicos que coloca maior dificuldade no diagnóstico de hipersensibilidade, uma vez que grande parte dos testes não se encontram validados. Os métodos de avaliação da expressão de moléculas da superfície dos basófilos por citometria de fluxo após activação induzida por fármacos têm sido referidos como uma possível mais‑valia no diagnóstico destas reacções. Objectivo: Avaliar a eficácia diagnóstica do teste de activação de basófilos (TAB) em doentes com história de hipersensibilidade imediata ao diclofenac. População e métodos: 17 doentes (M: 4, F: 13; idade média: 42,9 ± 13,7 anos) com história de hipersensibilidade ao diclofenac. Como controlo foram incluídos 11 voluntários saudáveis (M: 4, F: 7; idade média: 44,3 ± 18,7 anos) sem história de hipersensibilidade a AINEs. Foi realizado o TAB com diferentes abordagens na identificação da população de basófilos: Flow2 CAST® que utiliza CCR3, Basotest® - IgE, e o Basotest® modificado - HLA‑DR‑/CD123+. A avaliação da activação foi efectuada por citometria de fluxo, quantificando a expressão de novo de CD63 e up regulation de CD203c. Resultados: Foram comparados diferentes marcadores usados na identificação de basófilos...