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1.
Elife ; 132024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38251732

RESUMO

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single-particle cryo electron microscopy, cryo electron tomography, and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes in SlaA play important roles in S-layer assembly.


Assuntos
Sulfolobus acidocaldarius , Sulfolobus acidocaldarius/metabolismo , Archaea , Bactérias , Parede Celular
2.
J Am Acad Audiol ; 31(7): 496-505, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32119819

RESUMO

BACKGROUND: Pediatric hearing instrument fitting is optimally performed with individually obtained real-ear-to-coupler difference (RECD) measurements. If these measurements cannot be obtained, predicted values based on age are used. Recent evidence obtained from children aged 3-11 years suggests that head circumference (HC) may be a viable alternative or addition to age for use in RECD prediction. PURPOSE: The purpose of the present study was to determine if HC can be used to predict RECDs in infants, children, and adults. RESEARCH DESIGN: A correlational design was used. HC and RECD values were measured in all participants. STUDY SAMPLE: Participants were 278 North American infants and children (136 males and 142 females) aged 1.6 months to 11 years and 109 adults (42 males and 67 females) aged 18 years to 83 years. DATA COLLECTION AND ANALYSIS: After otoscopic inspection and immittance measurements were performed to assess candidacy for inclusion in the study, HC was measured twice for all participants and a single RECD measure was obtained for each participant at twelve frequencies (250 through 12500 Hz). The reliability of HC measurements was assessed with an intraclass correlation analysis. Linear regression analyses were performed with age and HC as predictor variables and RECDs as the dependent variable. RESULTS: Analysis indicated good reliability of the HC measurement. The relationships between RECD and HC were comparable with the relationships between RECD and age. Combining HC and age did not improve predictive accuracy. CONCLUSIONS: HC can be used in children and adults as an alternative metric in the prediction of RECDs when individual RECDs cannot be obtained.


Assuntos
Auxiliares de Audição , Adulto , Cefalometria , Criança , Orelha , Feminino , Audição , Humanos , Lactente , Masculino , Reprodutibilidade dos Testes
3.
J Am Acad Audiol ; 25(9): 823-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25405838

RESUMO

BACKGROUND: Ideally, individual real-ear-to-coupler difference (RECD) measurements are obtained for pediatric hearing instrument-fitting purposes. When RECD measurements cannot be obtained, age-related average RECDs based on typically developing North American children are used. Evidence suggests that these values may not be appropriate for populations of children with retarded growth patterns. PURPOSE: The purpose of this study was to determine if another metric, such as head circumference, height, or weight, can be used for prediction of RECDs in children. RESEARCH DESIGN: Design was a correlational study. For all participants, RECD values in both ears, head circumference, height, and weight were measured. STUDY SAMPLE: The sample consisted of 68 North American children (ages 3-11 yr). DATA COLLECTION AND ANALYSIS: Height, weight, head circumference, and RECDs were measured and were analyzed for both ears at 500, 750, 1000, 1500, 2000, 3000, 4000, and 6000 Hz. A backward elimination multiple-regression analysis was used to determine if age, height, weight, and/or head circumference are significant predictors of RECDs. RESULTS: For the left ear, head circumference was retained as the only statistically significant variable in the final model. For the right ear, head circumference was retained as the only statistically significant independent variable at all frequencies except at 2000 and 4000 Hz. At these latter frequencies, weight was retained as the only statistically significant independent variable after all other variables were eliminated. CONCLUSIONS: Head circumference can be considered as a metric for RECD prediction in children when individual measurements cannot be obtained. In developing countries where equipment is often unavailable and stunted growth can reduce the value of using age as a metric, head circumference can be considered as an alternative metric in the prediction of RECDs.


Assuntos
Desenho de Equipamento , Audição , Cefalometria , Criança , Pré-Escolar , Orelha/anatomia & histologia , Feminino , Auxiliares de Audição , Testes Auditivos , Humanos , Crânio
4.
J Immunol ; 182(5): 3121-30, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19234209

RESUMO

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4(+) T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition of EAE was due to reduced priming of autoreactive CD4(+) T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Receptores CCR6/antagonistas & inibidores , Receptores CCR6/fisiologia , Sequência de Aminoácidos , Animais , Quimiocina CCL20/biossíntese , Quimiocina CCL20/genética , Quimiocina CCL20/fisiologia , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Vasos Linfáticos/imunologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Receptores CCR6/biossíntese , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia
5.
Brain Pathol ; 18(4): 504-16, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18422759

RESUMO

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores Imunológicos/farmacologia , Receptores CXCR3/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Transferência Adotiva , Animais , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/fisiopatologia , Quimiocina CXCL11/antagonistas & inibidores , Quimiocina CXCL11/genética , Quimiocina CXCL11/imunologia , Quimiocina CXCL12/antagonistas & inibidores , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Quimiocinas/agonistas , Quimiocinas/genética , Quimiocinas/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Fatores Imunológicos/uso terapêutico , Terapia de Imunossupressão/métodos , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/farmacologia , Receptores CXCR3/imunologia , Receptores CXCR4/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento
6.
Biochemistry ; 45(25): 7882-8, 2006 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-16784240

RESUMO

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.


Assuntos
Dissulfetos/química , Interleucina-8/química , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Substituição de Aminoácidos , Dimerização , Interleucina-8/metabolismo , Ressonância Magnética Nuclear Biomolecular
7.
Biochemistry ; 44(46): 15414-21, 2005 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-16285746

RESUMO

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.


Assuntos
Proteína de Transporte de Acila/síntese química , Proteína de Transporte de Acila/metabolismo , Malonatos/metabolismo , Policetídeo Sintases/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Apoproteínas/síntese química , Apoproteínas/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Espectrometria de Massas por Ionização por Electrospray , Streptomyces coelicolor/enzimologia
8.
Am J Pathol ; 165(6): 2157-66, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15579457

RESUMO

The foreign body reaction (FBR) develops in response to the implantation of almost all biomaterials and can be detrimental to their function. The formation of foreign body giant cells (FBGC), which damage the surface of biomaterials, is considered a hallmark of this reaction. FBGC derive from blood-borne monocytes that enter the implantation site after surgery in response to the release of chemotactic signals. In this study, we implanted biomaterials subcutaneous (s.c.) in mice that lack the monocyte chemoattractant CC chemokine ligand 2 (CCL2) and found that biomaterials were encapsulated despite reduced FBGC formation. The latter was due to compromised macrophage fusion rather than migration. Consistent with the reduction in FBGC formation, biodegradable biomaterials sustained reduced damage in CCL2-null mice. Furthermore, blockade of CCL2 function by localized gene delivery in wild-type mice hindered FBGC formation, despite normal monocyte recruitment. The requirement for CCL2 in fusion was confirmed by the ability of both a CCL2 inhibitory peptide and an anti-CCL2 Ab to reduce FBGC formation from peripheral blood monocytes in an in vitro assay. Our findings demonstrate a previously unreported involvement of CCL2 in FBGC formation, and suggest that FBGC are not the primary determinants of capsule formation in the FBR.


Assuntos
Quimiocina CCL2/fisiologia , Células Gigantes de Corpo Estranho/metabolismo , Células Gigantes de Corpo Estranho/patologia , Macrófagos/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Materiais Biocompatíveis/administração & dosagem , Fusão Celular , Movimento Celular , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocinas CC/metabolismo , Feminino , Ligantes , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Fragmentos de Peptídeos/farmacologia
9.
Lab Invest ; 84(11): 1501-11, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15448707

RESUMO

While chemokines are clearly important in the generation of protective immunity, the role of individual chemokines in the control of bacterial infection is still poorly understood. In this study, we investigated the role of macrophage inflammatory protein (MIP)-3alpha/CCL20, a chemokine that attracts activated T and B lymphocytes and immature dendritic cells, in host responses to bacterial infection. CCL20 production was induced in subcutaneous tissue in the BALB/c mouse in response to Salmonella enteritidis, Staphylococcus aureus and zymosan, with S. enteritidis being the most potent. S. enteritidis induced CCL20 production in the spleen following either oral administration or injection into the peritoneal cavity. In contrast, no increase was observed in the Peyer's patches. In this model, following intraperitoneal injection, dose-dependent colonization of the spleen and Peyer's patches by S. enteritidis, expression of IFNgamma and IL-4, and production of antibodies against the S. enteritidis surface antigen SefA were observed. Prior treatment with neutralizing antibodies against CCL20 enhanced bacterial dissemination to the spleen and Peyer's patches and strongly biased the IFNgamma/IL-4 ratio towards a type 2 profile in the spleen, while the humoral response was unaffected. In contrast, treatment with neutralizing anti-MIP-1alpha/CCL3 antibodies enhanced the bacterial burden in the Peyer's patches but not in the spleen, had no significant effect on the cytokine ratio, but significantly inhibited anti-SefA production. Together, these results demonstrate an important role for CCL20 in the control of bacterial infection and more specifically in the regulation of cell-mediated immunity against intracellular bacteria such as S. enteritidis.


Assuntos
Quimiocinas CC/fisiologia , Proteínas Inflamatórias de Macrófagos/fisiologia , Infecções por Salmonella/microbiologia , Salmonella enteritidis/crescimento & desenvolvimento , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Quimiocina CCL20 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/imunologia , Contagem de Colônia Microbiana , Relação Dose-Resposta Imunológica , Feminino , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Interferon gama/biossíntese , Interleucina-4/biossíntese , Proteínas Inflamatórias de Macrófagos/imunologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Cavidade Peritoneal/microbiologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/microbiologia , Infecções por Salmonella/imunologia , Salmonella enteritidis/imunologia , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia
10.
Protein Sci ; 13(8): 2022-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15273303

RESUMO

CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a beta-bulge in beta-strand 1 that distorts the dimerization interface used by other CXC chemokines.


Assuntos
Quimiocinas CXC/química , Receptores de Quimiocinas/química , Quimiocina CXCL11 , Quimiocinas CXC/metabolismo , Dimerização , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo
11.
J Biol Chem ; 279(39): 40276-82, 2004 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-15231820

RESUMO

Chemokines constitute a group of over 40 secreted peptides that are important for the control of leukocyte migration both during homeostasis and inflammation. Recent studies have implicated the ligands CCL19 and CCL21 and their receptor, CCR7, in the specific migration of naïve lymphocytes and mature dendritic cells to secondary lymphoid organs during immune homeostasis. However, the role that these molecules play during immune priming is not well understood. In this study, using CCL19((8-83)), a novel N-terminal truncation mutant, we have investigated the role of CCL19 in a primary allogeneic immune response, a response of particular relevance to transplant rejection. This antagonist specifically inhibited wild type CCL19-induced chemotaxis and intracellular calcium mobilization without affecting that of CCL21. The treatment of mice with CCL19((8-83)) did not globally inhibit the recruitment of cells into lymph nodes; however, it inhibited the generation of cytotoxic T lymphocytes toward allogeneic dendritic cells. This is the first evidence that CCL19 plays a role in immune priming.


Assuntos
Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/metabolismo , Linfócitos T Citotóxicos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Movimento Celular , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas/metabolismo , Quimiotaxia , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Sistema Imunitário , Inflamação , Leucócitos/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR7 , Receptores de Quimiocinas/metabolismo , Baço/citologia , Fatores de Tempo
12.
Rheumatology (Oxford) ; 43(1): 39-42, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14566030

RESUMO

OBJECTIVE: To investigate the effect of targeting the chemotaxis of monocytes and polymorphonuclear monocytes (PMNs) in situ in MRL-Faslpr arthritis. METHODS: MRL-Faslpr mice were injected intradermally with complete Freund's adjuvant and cellular infiltration into the joint was monitored. Once clinical disease developed, the animals received one of three treatments: MCP-1(9-76); MCP-1(9-76) plus Gro-alpha(8-73); or control peptide, MCP-1 Ala. The bimalleolar ankle width was measured for 11 days and histological examination of the joints was then assessed. RESULTS: Cellular infiltration started after the onset of ankle swelling, and increased progressively. The incidence of swelling and the histopathology was reduced after day 6 of treatment in the MCP-1(9-76)-treated mice. Mice treated with the two antagonists MCP-1(9-76) and Gro-alpha(8-73) displayed a further significant reduction in disease parameters. CONCLUSION: Treatment after disease onset with chemotactic antagonists for monocytes and PMNs significantly alleviated both the swelling and the histopathology seen in arthritis, suggesting that chemokine antagonists are an effective anti-inflammatory therapy.


Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Quimiocinas/antagonistas & inibidores , Animais , Artrite Experimental/patologia , Quimiocina CCL2/uso terapêutico , Quimiocina CXCL1 , Quimiocinas/uso terapêutico , Fatores Quimiotáticos/uso terapêutico , Quimioterapia Combinada , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Articulações/patologia , Masculino , Camundongos , Camundongos Mutantes , Modelos Animais , Monócitos/patologia , Neutrófilos/patologia , Fragmentos de Peptídeos/uso terapêutico
13.
Nat Neurosci ; 6(10): 1064-71, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14502291

RESUMO

The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell-derived factor 1 alpha (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5-67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.


Assuntos
Complexo AIDS Demência/enzimologia , Quimiocinas CXC/toxicidade , Metaloproteinase 2 da Matriz/metabolismo , Degeneração Neural/enzimologia , Neurotoxinas/toxicidade , Complexo AIDS Demência/etiologia , Complexo AIDS Demência/fisiopatologia , Animais , Anticorpos/farmacologia , Astrócitos/metabolismo , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/enzimologia , Encefalite/fisiopatologia , Inibidores Enzimáticos/farmacologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Macrófagos/enzimologia , Macrófagos/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Neostriado/efeitos dos fármacos , Neostriado/patologia , Neostriado/fisiopatologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/virologia , Neurotoxinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade
14.
J Virol ; 77(15): 8588-92, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12857930

RESUMO

Viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus 68 gene M3 encodes a chemokine binding protein (vCKBP-3) which has no sequence similarity to chemokine receptors but inhibits chemokine receptor binding and activity. We have used a panel of CXCL8 analogs to identify the structural requirements for CXCL8 to bind to vCKBP-3 in a scintillation proximity assay. Our data suggest that vCKBP-3 acts by mimicking the binding of chemokine receptors to CXCL8.


Assuntos
Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Gammaherpesvirinae/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Quimiocinas CXC/genética , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica
15.
J Immunol ; 170(12): 6298-306, 2003 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12794163

RESUMO

Chemokines are a family of cytokines that exhibit selective chemoattractant properties for target leukocytes and play a significant role in leukocyte migration. In this study, we have investigated the role of the C-C chemokine, macrophage inflammatory protein (MIP)-3alpha/CC chemokine ligand 20, in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of T cell-dependent inflammation. Expression in the CNS of MIP-3alpha, as determined by RT-PCR, increased in a time-dependent manner such that peak expression correlated with peak clinical disease. Similarly, levels of immunoreactive MIP-3alpha in the draining lymph nodes increased up to 10-fold 9 days postimmunization and remained elevated for up to 21 days postimmunization. The increased production of MIP-3alpha coincided with onset of clinical disease. Treatment of mice with specific neutralizing anti-MIP-3alpha Abs significantly reduced the severity of both clinical EAE and neuroinflammation by inhibiting the sensitization of lymphocytes to the specific Ag and release of lymphocytes from the draining lymph nodes. In contrast, adoptive transfer experiments indicated that MIP-3alpha was not essential for the effector phase of EAE. Together, these data demonstrate that MIP-3alpha plays a critical role in the sensitization phase of EAE.


Assuntos
Quimiocinas CC/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Imunização , Proteínas Inflamatórias de Macrófagos/fisiologia , Receptores de Quimiocinas , Medula Espinal/imunologia , Linfócitos T/imunologia , Animais , Inibição de Migração Celular , Movimento Celular/imunologia , Células Cultivadas , Quimiocina CCL20 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/biossíntese , Quimiocinas CC/imunologia , Progressão da Doença , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Soros Imunes/farmacologia , Imunização/métodos , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Proteínas Inflamatórias de Macrófagos/antagonistas & inibidores , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas Inflamatórias de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Proteína Proteolipídica de Mielina/administração & dosagem , Proteína Proteolipídica de Mielina/imunologia , Receptores CCR6 , Medula Espinal/metabolismo , Medula Espinal/patologia , Linfócitos T/citologia , Linfócitos T/patologia , Regulação para Cima/imunologia
16.
Blood ; 102(3): 789-94, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12689946

RESUMO

Eotaxin-3 (CCL26) belongs to the group of CC chemokines that attract eosinophils, basophils, and Th2 lymphocytes. Like eotaxin (CCL11) and eotaxin-2 (CCL24), eotaxin-3 mediates its activity through CCR3. Here we show that eotaxin-3 also binds to CCR2 on monocytes and CCR2-transfected cells. In contrast to monocyte chemotactic protein 1 (MCP-1; CCL2), eotaxin-3 does not trigger intracellular calcium mobilization, enzyme release, or phosphorylation of the mitogen-activated protein (MAP) kinase ERK and induces a weak chemotaxis in monocytes. Instead, eotaxin-3 inhibits MCP-1-mediated responses, thus acting as a natural antagonist for CCR2. This study also demonstrates that eotaxin-3 promotes active movement of monocytes away from a gradient of eotaxin-3 in vitro. This repellent effect is amplified when an additional gradient of MCP-1 is applied, demonstrating that the 2 mechanisms are synergistic. Eotaxin-3 effects on monocytes are largely abolished when cells are pretreated with MCP-1 or CCR2 antagonists. Like MCP-1-mediated migration, repulsion is sensitive to Bordetella pertussis toxin, indicating the involvement of Gi protein-coupled receptors. However, using transfected cells expressing CCR2 we could not detect F-actin formation or an active movement away induced by eotaxin-3, suggesting that either expression of a single receptor type is not sufficient to mediate cell repulsion or that the used transfected cell lines lack additional interaction molecules that are required for reverse migration. Eotaxin-3 was expressed by vascular endothelial cells and was essential for endothelial transmigration of eosinophils. Our data provide a mechanism by which 2 chemokine gradients that are oriented in opposite directions could cooperate in efficiently driving out monocytes from blood vessels into tissue.


Assuntos
Quimiocinas CC/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Monócitos/citologia , Receptores de Quimiocinas/antagonistas & inibidores , Cálcio/metabolismo , Quimiocina CCL26 , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacologia , Humanos , Inflamação/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , Ligação Proteica , Receptores CCR2 , Transfecção
17.
J Mol Biol ; 327(2): 329-34, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12628240

RESUMO

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.


Assuntos
Quimiocinas CXC/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Motivos de Aminoácidos , Ligação Competitiva , Quimiocina CXCL12 , Quimiocina CXCL2 , Quimiocinas CXC/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Monocinas/química , Monocinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos , Ligação Proteica , Receptores CXCR4/química , Relação Estrutura-Atividade
18.
Am J Pathol ; 162(4): 1183-90, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12651610

RESUMO

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.


Assuntos
Neoplasias da Mama/patologia , Catepsina D/metabolismo , Quimiocinas CC/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Catepsina D/genética , Quimiocina CCL21 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/química , Quimiocinas CC/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Cinética , Proteínas Inflamatórias de Macrófagos/química , Proteínas Inflamatórias de Macrófagos/genética , Dados de Sequência Molecular , Especificidade por Substrato , Células Tumorais Cultivadas
19.
Proc Natl Acad Sci U S A ; 100(4): 1885-90, 2003 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-12571364

RESUMO

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1beta/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.


Assuntos
Quimiocinas/fisiologia , Glicosaminoglicanos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Biopolímeros , Células CHO , Quimiocinas/metabolismo , Quimiotaxia de Leucócito , Cricetinae , Primers do DNA , Feminino , Glicosaminoglicanos/química , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Cavidade Peritoneal/citologia , Proteínas Recombinantes/metabolismo
20.
J Biol Chem ; 278(13): 10928-33, 2003 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-12554737

RESUMO

The chemokine-like, secreted protein product of the U83 gene from human herpesvirus 6, here named vCCL4, was chemically synthesized to be characterized in a complete library of the 18 known human chemokine receptors expressed individually in stably transfected cell lines. vCCL4 was found to cause calcium mobilization as efficiently as the endogenous chemokine ligand CCL2 through the CCR2 receptor, whereas the virally encoded chemokine did not affect any of the other 17 human chemokine receptors tested. Mutual cross-desensitization between CCL2 and vCCL4 was demonstrated in the CCR2-transfected cells. The affinity of vCCL4 for the CCR2 receptor was 79 nm as determined in competition binding against radioactively labeled CCL2. In the murine pre-B lymphocyte cell line L1.2 stably transfected with the CCR2 receptor, vCCL4 acted as a relatively low potency but highly efficacious chemoattractant being equally or more efficacious in causing cell migration than CCL2 and CCL7 and considerably more efficacious than CCL8 and CCL13. It is concluded that human herpesvirus 6 encodes a highly selective and efficacious CCR2 agonist, which will attract CCR2 expressing cells, for example macrophages and monocytes, conceivably for the virus to infect and to establish latency in. It is suggested that vCCL4 during reactivation of the virus in for example monocyte-derived microglia could perhaps be involved in the pathogenesis of the CCR2-dependent disease, multiple sclerosis.


Assuntos
Quimiocinas/genética , Herpesvirus Humano 6/genética , Receptores de Quimiocinas/agonistas , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Cricetinae , Genes Virais , Dados de Sequência Molecular , Receptores CCR2 , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
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