RESUMO
Background: International guidelines for neuroprotection following out-of-hospital cardiac arrest (OHCA) recommend fever prevention ahead of routine temperature management. This study aimed to identify any effect of changing from targeted temperature management to fever prevention on neurological outcome following OHCA. Methods: A retrospective observational cohort study was conducted of consecutive admissions to an ICU at a tertiary OHCA centre. Comparison was made between a period of protocolised targeted temperature management (TTM) to 36 °C and a period of fever prevention. Results: Data were available for 183 patients. Active temperature management was administered in 86/118 (72%) of the TTM cohort and 20/65 (31%) of the fever prevention group. The median highest temperature prior to the start of temperature management was significantly lower in the TTM group at 35.6 (IQR 34.9-36.2) compared to 37.9 °C (IQR 37.7-38.2) in the fever prevention group (adjusted p < 0.001).There was no difference in the proportion of patients discharged with Cerebral Performance Category 1 or 2 between the groups (42% vs. 40%, p = 0.88). Patients in the fever prevention group required a reduced duration of noradrenaline (36 vs. 46 h, p = 0.03) and a trend towards a reduced duration of propofol (37 vs. 56 h, p = 0.06).In unadjusted analysis, use of active temperature management (irrespective of group) appeared to be associated with decreased risk of poor outcome (OR = 0.43, 95% CI 0.23-0.78) but after adjustment for patient age, presenting rhythm, witnessed arrest and duration of CPR, this was no longer significant (OR = 0.93, 95% CI 0.37-2.31, p = 0.88). Conclusion: Switching from TTM to fever prevention following OHCA was associated with similar rates of neurological outcomes, with a possible decrease in sedation and vasopressor requirements.
RESUMO
Lymph-borne, soluble factors (e.g., chemokines and others) influence lymphocyte recirculation and endothelial phenotype at high endothelial venules (HEVs) in lymph node cortex. Yet the route lymph-borne soluble molecules travel from the subcapsular sinus to the HEVs is unclear. Therefore, we injected subcutaneously into mice and rats a wide variety of fluorophore-labeled, soluble molecules and examined their distribution in the draining lymph nodes. Rather than percolating throughout the draining lymph node, all molecules, including microbial lipopolysaccharide, were very visible in the subcapsular and medullary sinuses but were largely excluded from the cortical lymphocyte microenvironments. Exclusion prevailed even during the acute lymph node enlargement accompanying viral infection. However, low molecular mass (MW) molecules, including chemokines, did gain entry into the cortex, but in a very defined manner. Low MW, fluorophore-labeled molecules highlighted the subcapsular sinus, the reticular fibers, and the abluminal and luminal surfaces of the associated HEVs. These low MW molecules were in the fibers of the reticular network, a meshwork of collagen fibers ensheathed by fibroblastic reticular cells that connects the subcapsular sinus floor and the HEVs by intertwining with their basement membranes. Thus, low MW, lymph-borne molecules, including chemokines, traveled rapidly from the subcapsular sinus to the HEVs using the reticular network as a conduit.
Assuntos
Quimiocinas/metabolismo , Quimiotaxia de Leucócito/fisiologia , Linfonodos/fisiologia , Linfa/fisiologia , Sistema Linfático/fisiologia , Linfócitos T/fisiologia , Animais , Transporte Biológico , Feminino , Centro Germinativo , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Distribuição Tecidual , VacíniaRESUMO
The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell-attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.
Assuntos
Quimiocinas CC/fisiologia , Dermatite/imunologia , Linfócitos/fisiologia , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/fisiologia , Transferência Adotiva , Animais , Movimento Celular , Quimiocina CCL17 , Feminino , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Receptores CCR10 , Receptores CCR4RESUMO
The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.
Assuntos
Quimiocina CCL2 , Quimiocina CCL5 , Glomerulonefrite/etiologia , Animais , Colágeno/biossíntese , Colágeno/genética , Modelos Animais de Doenças , Progressão da Doença , Fibrose/etiologia , Imuno-Histoquímica , Rim/patologia , Camundongos , Proteinúria , RNA Mensageiro/análiseRESUMO
Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.
Assuntos
Cisteína Endopeptidases/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Apoptose , Caspase 1 , Granzimas , Humanos , Interleucina-1/metabolismo , Dados de Sequência Molecular , Precursores de Proteínas/metabolismoRESUMO
Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
Assuntos
Quimiocinas CC , Células Dendríticas/imunologia , Pulmão/imunologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores de Quimiocinas , Receptores de Citocinas/genética , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL20 , Clonagem Molecular , Primers do DNA/genética , Células Dendríticas/metabolismo , Expressão Gênica , Humanos , Pulmão/metabolismo , Proteínas Inflamatórias de Macrófagos/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR6 , Receptores de Citocinas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.
Assuntos
Eosinófilos/imunologia , Interleucina-5/imunologia , Células Cultivadas , Humanos , Interleucina-5/genética , Mutação , Receptores de Interleucina/imunologia , Receptores de Interleucina-5 , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Quimiocina CCL5/análogos & derivados , HIV-1/efeitos dos fármacos , Receptores CCR5/metabolismo , Animais , Transporte Biológico , Células CHO , Quimiocina CCL5/farmacologia , Cricetinae , Regulação para Baixo , Endocitose , Endossomos/metabolismo , HumanosRESUMO
CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.
Assuntos
Quimiocinas CC/metabolismo , Receptores de Quimiocinas/metabolismo , Choque Séptico/imunologia , Linfócitos T/imunologia , Animais , Sequência de Bases , Quimiocina CCL17 , Quimiocina CCL22 , Primers do DNA/genética , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Receptores CCR4 , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Choque Séptico/patologia , Choque Séptico/prevenção & controle , Células Th2/imunologiaRESUMO
The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1alpha, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.
Assuntos
Quimiocinas CC/fisiologia , Hipersensibilidade/imunologia , Inflamação/imunologia , Pulmão/imunologia , Animais , Anticorpos/imunologia , Asma/fisiopatologia , Quimiocina CCL11 , Quimiocina CCL4 , Quimiocina CCL5/farmacologia , Quimiocinas CC/antagonistas & inibidores , Fatores Quimiotáticos de Eosinófilos/farmacologia , Citocinas/farmacologia , Modelos Animais de Doenças , Imuno-Histoquímica , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Pulmão/citologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos , Proteínas Quimioatraentes de Monócitos/farmacologia , Ovalbumina/imunologia , RNA Mensageiro/metabolismoRESUMO
Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12-24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.
Assuntos
Asma/fisiopatologia , Quimiocinas CC , Fatores Quimiotáticos de Eosinófilos/biossíntese , Citocinas/biossíntese , Eosinófilos/fisiologia , Animais , Células da Medula Óssea , Líquido da Lavagem Broncoalveolar/química , Quimiocina CCL11 , Citocinas/análise , Dexametasona/farmacologia , Feminino , Cobaias , Interleucina-5/fisiologia , Pulmão/patologia , Masculino , Albumina Sérica/análiseRESUMO
The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1alpha, and MIP-1beta. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH(2)-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect. Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6-9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES-induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein-coupled receptor trafficking through the recycling endosome compartment.
Assuntos
Endocitose , Receptores CCR5/metabolismo , Animais , Fármacos Anti-HIV/metabolismo , Células CHO , Compartimento Celular , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/metabolismo , Cricetinae , Humanos , LigantesRESUMO
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein-coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Galphai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.
Assuntos
Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/fisiologia , Quimiotaxia/fisiologia , Proteínas do Leite , Transdução de Sinais/fisiologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteínas de Ligação ao GTP/metabolismo , Humanos , Janus Quinase 1 , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores CCR5/genética , Receptores CCR5/fisiologia , Fator de Transcrição STAT5 , Transativadores/metabolismo , Transfecção , Tirosina/metabolismoRESUMO
The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.
Assuntos
HIV-1/efeitos dos fármacos , Macrófagos/virologia , Receptores de Quimiocinas , Receptores de Citocinas/antagonistas & inibidores , Receptores de HIV/antagonistas & inibidores , Linfócitos T/virologia , Animais , Ligação Competitiva , Antígenos CD4/metabolismo , Gatos , Linhagem Celular , Células Cultivadas , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Quimiotaxia de Leucócito , HIV-1/fisiologia , Células HeLa , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Receptores CCR5 , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Linfócitos T/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Henry Matthew was appointed a consultant in the Royal Infirmary of Edinburgh in 1955, by which time he was a highly regarded general physician with an interest in cardiology. In 1964 he agreed, almost certainly reluctantly, to head the recently designated Regional Poisoning Treatment Centre, which he did until his retirement ten years later. Matthew quickly established himself as an authority in clinical toxicology, mainly from an unrivalled experience of treating poisoned patients, day-in and day-out, but also by publishing original research, letters and books. Such were his contributions that he is regarded as the father of clinical toxicology.
Assuntos
Toxicologia/história , História do Século XX , História do Século XXI , EscóciaRESUMO
Despite their key role in inflammation, the apparent redundancy in the chemokine system is often cited as an argument against probing chemokines as therapeutic targets for inflammation. However, this in vitro redundancy frequently does not translate to the in vivo situation, as exemplified by the use of specific receptor antagonists, ligand neutralizing or receptor blocking antibodies and gene-deleted mice in models of human disease. Specificity may be conferred onto the chemokine system by fine-tuning of responses both temporally and spatially through their highly specific interactions with glycosaminoglycans (GAGs). In this survey, we present evidence for specificity in the interaction and introduce emerging technologies that enable detailed assessment of protein-GAG interactions. Finally, we address the issue of exploitation of this interaction for therapeutic advantage.
Assuntos
Quimiocinas/metabolismo , Sistemas de Liberação de Medicamentos , Glicosaminoglicanos/metabolismo , Doenças do Sistema Imunitário/tratamento farmacológico , Glicosaminoglicanos/química , Glicosaminoglicanos/classificação , Humanos , Modelos MolecularesRESUMO
Selective eosinophil recruitment into tissues is a characteristic feature of allergic diseases. Chemokines are effective leukocyte chemoattractants and may play an important role in mediating eosinophil recruitment in various allergic conditions in man. Here, we describe a novel mouse model of eosinophil recruitment in which we have compared the in vivo chemoattractant activity of different C-C chemokines. Furthermore, we describe the use of antibodies to chemokines and receptor blockade to address the endogenous mechanisms involved in eosinophil recruitment in a late-phase allergic reaction in mouse skin. Intradermal injection of mEotaxin and mMIP-1alpha, but not mMCP-1, mRANTES, mMCP-5, or mMIP-1beta, induced significant 111In-eosinophil recruitment in mouse skin. Significant 111In-eosinophil recruitment was also observed in an active cutaneous anaphylactic reaction. Pretreatment of skin sites with antieotaxin antiserum, but not an antiMIP-1alpha antibody, suppressed 111In-eosinophil recruitment in this delayed-onset allergic reaction. Similarly, desensitization of the eosinophil eotaxin receptor CCR3 with mEotaxin, or blockade of the receptor with metRANTES, significantly inhibited 111In-eosinophil recruitment in the allergic reaction. These results demonstrate an important role for endogenous eotaxin in mediating the 111In-eosinophil recruitment in allergic inflammation, and suggest that blockade of the CCR3 receptor is a valid strategy to inhibit eosinophil migration in vivo.
Assuntos
Quimiocinas CC/farmacologia , Quimiotaxia de Leucócito , Eosinófilos/imunologia , Hipersensibilidade/imunologia , Pele/imunologia , Anafilaxia/imunologia , Animais , Quimiocina CCL11 , Fatores Quimiotáticos de Eosinófilos/metabolismo , Complemento C5a/farmacologia , Citocinas/metabolismo , Feminino , Hipersensibilidade Tardia/imunologia , Leucotrieno B4/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Fator de Ativação de Plaquetas/farmacologia , Receptores CCR3 , Receptores de Quimiocinas/metabolismoRESUMO
We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.
Assuntos
Regulação Fúngica da Expressão Gênica , Manose-6-Fosfato Isomerase/genética , Manose/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Fúngicos , Análise Mutacional de DNA , Dolicol Monofosfato Manose/metabolismo , Teste de Complementação Genética , Glicosilação , Guanosina Difosfato Manose/biossíntese , Íntrons/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Mapeamento por Restrição , Transcrição GênicaRESUMO
Interleukin-6 (IL-6) is a proinflammatory cytokine which also acts as a growth factor for some murine hybridomas (7TD1) or human myelomas (U266). We demonstrate that elevation of cAMP cellular content inhibits IL-6-stimulated cell growth, by blocking cells mainly in G1 phase. This inhibition is associated with increased expression of the Fos family protein Fra-2. Treatment of cells with 8Br-cAMP results in increased DNA-binding activity of two distinct AP-1 complexes; JunD/Fra-2 and JunB/Fra-2, and also in elevated AP-1 transactivation. When 8Br-cAMP is withdrawn from the medium, cells enter S phase and Fra-2 protein levels and AP-1 DNA-binding activity decrease to their basal value indicating that a temporally correlation exists between the 8Br-cAMP-mediated induction of JunD/Fra-2 AP-1 complex and the 7TD1 and U266 cell growth inhibition.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular , Colforsina/farmacologia , DNA/metabolismo , Antígeno 2 Relacionado a Fos , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-fos/biossíntese , Ativação TranscricionalRESUMO
High-density lipoprotein (HDL) subclasses 2 and 3 prepared by density gradient ultracentrifugation have been further fractionated by immunoaffinity chromatography using antibody affinity gels targetting the major HDL apolipoproteins, A-I and A-II. Fractions containing A-I without A-II (AI w/o AII) and A-I with A-II (AI w AII) were isolated from both density ranges. Whereas there were similar concentrations of the major subfraction (HDL3(AI w AII] in both males and females, the remaining subfractions were present in higher concentrations in females as compared to males, in the order HDL3 (AI w/o AII) less than HDL2(AI w AII) less than HDL2(AI w/o AII). The difference was most marked for HDL2 (AI w/o AII), where plasma concentrations in females were almost 3-fold greater than in males. Compositional analyses indicated that the plasma concentrations of the fractions, rather than their compositions, were the major determinants of male-female differences in HDL levels. In contrast, fractions defined by similar apolipoprotein criteria and isolated from different density subclasses (i.e., HDL2(AI w/o AII) vs. HDL3(AI w/o AII) and HDL2(AI w AII) vs. HDL3(AI w AII] showed major compositional differences. This is suggestive of distinct lipoprotein particles.