RESUMO
There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages.
Assuntos
Antiprotozoários/farmacologia , Quitosana/farmacologia , Leishmania major/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Anfotericina B/farmacologia , Animais , Quitosana/análogos & derivados , Meios de Cultura/química , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Concentração de Íons de Hidrogênio , Leishmania major/imunologia , Leishmania major/metabolismo , Leishmania mexicana/imunologia , Leishmania mexicana/metabolismo , Estágios do Ciclo de Vida/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Óxido Nítrico/metabolismo , Testes de Sensibilidade Parasitária , Pinocitose/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Células THP-1 , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.
Assuntos
Configuração de Carboidratos , Ativação do Complemento/efeitos dos fármacos , Complemento C2/metabolismo , Complemento C4/metabolismo , Via Clássica do Complemento/efeitos dos fármacos , Proteínas de Helminto/farmacologia , Fosforilcolina/metabolismo , Sítios de Ligação , Proteína C-Reativa/metabolismo , Convertases de Complemento C3-C5/metabolismo , Humanos , Ressonância de Plasmônio de SuperfícieRESUMO
No deficiency of human C-reactive protein (CRP), or even structural polymorphism of the protein, has yet been reported so its physiological role is not known. Here we show for the first time that CRP-deficient mice are remarkably susceptible to Streptococcus pneumoniae infection and are protected by reconstitution with isolated pure human CRP, or by anti-pneumococcal antibodies. Autologous mouse CRP is evidently essential for innate resistance to pneumococcal infection before antibodies are produced. Our findings are consistent with the significant association between clinical pneumococcal infection and non-coding human CRP gene polymorphisms which affect CRP expression. Deficiency or loss of function variation in CRP may therefore be lethal at the first early-life encounter with this ubiquitous virulent pathogen, explaining the invariant presence and structure of CRP in human adults.
Assuntos
Proteína C-Reativa/imunologia , Imunidade Inata , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Proteína C-Reativa/deficiência , Proteína C-Reativa/genética , Humanos , Camundongos , Camundongos Knockout , FenótipoRESUMO
Human C-reactive protein (CRP) binds to lipophosphoglycan (LPG), a virulence factor of Leishmania spp., through the repeating phosphodisaccharide region. We report here that both major components of promastigote secretory gel (PSG), the filamentous proteophosphoglycan (fPPG) and the secreted acid phosphatase (ScAP), are also ligands. CRP binding was mainly associated with the flagellar pocket when LPG deficient Leishmania mexicana parasites were examined by fluorescent microscopy, consistent with binding to secreted material. ScAP is a major ligand in purified fPPG from parasite culture as demonstrated by much reduced binding to a ScAP deficient mutant fPPG in plate binding assays and ligand blotting. Nevertheless, in sandfly derived PSG fPPG is a major component and the major CRP binding component. Previously we showed high avidity of CRP for LPG ligand required multiple disaccharide repeats. ScAP and fPPG only have short repeats but they retain high avidity for CRP revealed by surface plasmon resonance because they are found in multiple copies on the phosphoglycan. The fPPG from many species such as L. donovani and L. mexicana bound CRP strongly but L. tropica and L. amazonensis had low amounts of binding. The extent of side chain substitution of [-PO4-6Galß1-4Manα1-] disaccharides correlates inversely with binding of CRP. The ligand for the CRP on different species all had similar binding avidity as the half maximal binding concentration was similar. Since the PSG is injected with the parasites into host blood pools and phosphoglycans (PG) are known to deplete complement, we showed that CRP makes a significant contribution to the activation of complement by PSG using serum from naive donors.
Assuntos
Proteína C-Reativa , Leishmania , Humanos , Ligantes , Proteínas do Sistema Complemento , Transporte Biológico , DissacarídeosRESUMO
OBJECTIVE: Elevated serum levels of the acute-phase protein serum amyloid A (SAA) are a marker for active rheumatoid arthritis (RA), and SAA can also be found in the tissues of patients with active RA. Based on a number of studies with recombinant SAA (rSAA), the protein has been suggested to be a potent proinflammatory mediator that activates human neutrophils, but whether endogenous SAA shares these proinflammatory activities has not been directly addressed. The present study was undertaken to investigate whether SAA in the plasma of patients with RA possesses proinflammatory properties and activates neutrophils in a manner similar to that of the recombinant protein. METHODS: Neutrophil activation was monitored by flow cytometry, based on L-selectin shedding from cell surfaces. Whole blood samples from healthy subjects and from RA patients with highly elevated SAA levels were studied before and after stimulation with rSAA as well as purified endogenous SAA. RESULTS: Recombinant SAA potently induced cleavage of L-selectin from neutrophils and in whole blood samples. Despite highly elevated SAA levels, L-selectin was not down-regulated on RA patient neutrophils as compared with neutrophils from healthy controls. Spiking SAA-rich whole blood samples from RA patients with rSAA, however, resulted in L-selectin shedding. In addition, SAA purified from human plasma was completely devoid of neutrophil- or macrophage-activating capacity. CONCLUSION: The present findings show that rSAA is proinflammatory but that this activity is not shared by endogenous SAA, either when present in the circulation of RA patients or when purified from plasma during an acute-phase response.
Assuntos
Reação de Fase Aguda/imunologia , Artrite Reumatoide/imunologia , Leucócitos Mononucleares/imunologia , Ativação de Neutrófilo/imunologia , Proteína Amiloide A Sérica/imunologia , Reação de Fase Aguda/metabolismo , Artrite Reumatoide/sangue , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Selectina L/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/farmacologiaRESUMO
Antigens from Mycobacterium tuberculosis (M.tb), have been shown to stimulate human B cell responses to unrelated recall antigens in vitro. However, it is not known whether natural M.tb infection or whether vaccination with, Mycobacterium bovis BCG, has a similar effect. This study investigated the effects of M.tb infection and BCG vaccination on B cell responses to heterologous pathogen recall antigens. Antibodies against several bacterial and viral pathogens were quantified by ELISA in 68 uninfected controls, 62 individuals with latent TB infection (LTBI) and 107 active pulmonary TB (APTB) cases, and 24 recently BCG-vaccinated adolescents and naive controls. Antibody avidity was investigated using surface plasmon resonance and B cell ELISPOTs were used to measure plasmablast and memory B cell responses (MBC) in APTB cases and healthy donor controls. APTB was associated with higher levels of antibodies to respiratory syncytial virus and measles virus, compared to uninfected controls. BCG vaccination did not alter levels of antibodies against heterologous pathogens. Tetanus toxoid (TT)-specific antibody avidity was increased in APTB cases in comparison to uninfected individuals and the ratio of TT-specific plasmablasts to MBCs in the APTB cases was 7:1. M.tb infection is associated with increased antibody responses to heterologous pathogens in human subjects.
Assuntos
Antígenos Heterófilos/imunologia , Vacina BCG/imunologia , Tuberculose Latente/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Idoso , Afinidade de Anticorpos , Formação de Anticorpos , Linfócitos B/fisiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Toxoide Tetânico/imunologia , Adulto JovemRESUMO
The extent to which the acute phase response (APR) influences iron status indicators in chronic infections is not well documented. We investigated this relationship using reported recent fever and 2 acute phase proteins (APP), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). In a sample of 690 children matched on age and helminth infection status at baseline, we measured plasma for AGP, CRP, ferritin, transferrin receptor (TfR), and erythropoietin (EPO) and whole blood for hemoglobin (Hb) concentration, zinc protoporphyrin (ZPP), and malaria parasite density, and we obtained maternal reports of recent fever. We then examined the influence of the APR on each iron status indicator using regression analysis with Hb as the outcome variable. Ferritin was inversely related to Hb in the APR-unadjusted model. Adjusting for the APR using reported recent fever alone was not sufficient to reverse the inverse Hb-ferritin relationship. However, using CRP and/or AGP resulted in the expected positive relationship. The best fit model included reported recent fever, AGP and CRP (R(2) = 0.241; P < 0.001). The best fit Hb-ZPP, Hb-TfR, and Hb-EPO models included reported recent fever and AGP but not CRP (R(2) = 0.253, 0.310, and 0.292, respectively; P < 0.001). ZPP, TfR, and EPO were minimally influenced by the APR, whereas ferritin was immensely affected. Reported recent fever alone cannot be used as a marker for the APR. Either AGP or CRP is useful for adjusting if only 1 APP can be measured. However, AGP best predicted the APR in this population.
Assuntos
Helmintíase/epidemiologia , Ferro da Dieta/metabolismo , Ferro/metabolismo , Malária/epidemiologia , Proteínas de Fase Aguda/metabolismo , Apetite/fisiologia , Proteína C-Reativa/metabolismo , Ensaios Clínicos como Assunto , Eritropoetina/sangue , Feminino , Ferritinas/sangue , Helmintíase/sangue , Hemoglobinas/metabolismo , Humanos , Lactente , Malária/sangue , Masculino , Orosomucoide/metabolismo , Tanzânia/epidemiologiaRESUMO
During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.
Assuntos
Antígenos CD36/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteína Amiloide A Sérica/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Transdução de SinaisRESUMO
There is increasing evidence supporting a role for antibodies in protection against tuberculosis (TB), with functional antibodies being described in the latent state of TB infection. Antibody avidity is an important determinant of antibody-mediated protection. This study characterised the avidity of antibodies against Ag85A, an immunodominant Mycobacterium tuberculosis (M.tb) antigen and constituent of several anti-TB vaccine candidates, in individuals of varied M.tb infection status. Avidity of Ag85A specific antibodies was measured in 30 uninfected controls, 34 individuals with latent TB infection (LTBI) and 75 active pulmonary TB (APTB) cases, employing the more commonly used chaotrope-based dissociation assays, and surface plasmon resonance (SPR). Chaotrope-based assays indicated that APTB was associated with a higher antibody avidity index compared to uninfected controls [adjusted geometric mean ratio (GMR): 1.641, 95% confidence interval (CI): 1.153, 2.337, p = 0.006, q = 0.018] and to individuals with LTBI [adjusted GMR: 1.604, 95% CI: 1.282, 2.006, p < 0.001, q <0.001]. SPR assays showed that APTB was associated with slower dissociation rates, an indication of higher avidity, compared to uninfected controls (adjusted GMR: 0.796, 95% CI: 0.681, 0.932, p = 0.004, q = 0.012) and there was also weak evidence of more avid antibodies in the LTBI compared to the uninfected controls (adjusted GMR: 0.871, 95% CI: 0.763, 0.994, p = 0.041, q = 0.123). We found no statistically significant differences in anti-Ag85A antibody avidity between the APTB and LTBI groups. This study shows that antibodies of increased avidity are generated against a principle vaccine antigen in M.tb infected individuals. It would be important to determine whether TB vaccines are able to elicit a similar response. Additionally, more research is needed to determine whether antibody avidity is important in protection against infection and disease.
Assuntos
Anticorpos Antibacterianos/metabolismo , Afinidade de Anticorpos , Tuberculose Latente/imunologia , Mycobacterium tuberculosis/imunologia , Ressonância de Plasmônio de Superfície , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Coinfecção , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por HIV/complicações , Infecções por HIV/imunologia , Humanos , Lactente , Tuberculose Latente/complicações , Tuberculose Latente/diagnóstico , Masculino , Pessoa de Meia-Idade , Classe Social , Ressonância de Plasmônio de Superfície/métodos , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/diagnóstico , Adulto JovemRESUMO
QuantiFERON®-TB Gold in-tube (QFT-GIT) supernatants may be important samples for use in assessment of anti-tuberculosis (TB) antibodies when only limited volumes of blood can be collected and when a combination of antibody and cytokine measurements are required. These analytes, when used together, may also have the potential to differentiate active pulmonary TB (APTB) from latent TB infection (LTBI). However, few studies have explored the use of QFT-GIT supernatants for investigations of antibody responses. This study determined the correlation and agreement between anti-CFP-10 and anti-ESAT-6 antibody concentrations in QFT-GIT nil supernatant and serum pairs from 68 TB household contacts. We also explored the ability of Mycobacterium tuberculosis (M.tb) specific antibodies, or ratios of antibody to interferon gamma (IFN-γ) in QFT-GIT supernatants, to differentiate 97 APTB cases from 58 individuals with LTBI. Sputum smear microscopy was used to define APTB, whereas the QFT-GIT and tuberculin skin test were used to define LTBI. There were strong and statistically significant correlations between anti-CFP-10 and anti-ESAT-6 antibodies in unstimulated QFT-GIT supernatants and sera (r = 0.89; p<0.0001 for both), and no significant differences in antibody concentration between them. Anti-CFP-10 & anti-ESAT-6 antibodies differentiated APTB from LTBI with sensitivities of 88.7% & 71.1% and specificities of 41.4% & 51.7% respectively. Anti-CFP-10 antibody/M.tb specific IFN-γ and anti-ESAT-6 antibody/M.tb specific IFN-γ ratios had sensitivities of 48.5% & 54.6% and specificities of 89.7% and 75.9% respectively. We conclude that QFT-GIT nil supernatants may be used in the place of sera when measuring antibody responses, reducing blood volumes needed for such investigations. Antibodies in QFT-GIT nil supernatants on their own discriminate APTB from LTBI with high sensitivity but have poor specificity, whereas the reverse is true when antibodies are used in combination with M.tb specific cytokines. Further antibody and antibody/cytokine combinations need to be explored to achieve better diagnostic accuracy.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Tuberculose Latente/sangue , Tuberculose Pulmonar/sangue , Adolescente , Adulto , Idoso , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/isolamento & purificação , Formação de Anticorpos/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interferon gama/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Escarro/imunologia , Escarro/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.
Assuntos
Animais de Laboratório/imunologia , Animais Selvagens/imunologia , Camundongos/imunologia , Animais , Proteínas Sanguíneas/metabolismo , Citocinas/biossíntese , Fezes/química , Citometria de Fluxo , Haptoglobinas/metabolismo , Homeostase , Imunoglobulina A/análise , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunofenotipagem , Ativação Linfocitária , Subpopulações de Linfócitos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Componente Amiloide P Sérico/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
Immunoglobulin G (IgG) Fc N-glycosylation affects antibody-mediated effector functions and varies with inflammation rooted in both communicable and non-communicable diseases. Worldwide, communicable and non-communicable diseases tend to segregate geographically. Therefore, we studied whether IgG Fc N-glycosylation varies in populations with different environmental exposures in different parts of the world. IgG Fc N-glycosylation was analysed in serum/plasma of 700 school-age children from different communities of Gabon, Ghana, Ecuador, the Netherlands and Germany. IgG1 galactosylation levels were generally higher in more affluent countries and in more urban communities. High IgG1 galactosylation levels correlated with low total IgE levels, low C-reactive protein levels and low prevalence of parasitic infections. Linear mixed modelling showed that only positivity for parasitic infections was a significant predictor of reduced IgG1 galactosylation levels. That IgG1 galactosylation is a predictor of immune activation is supported by the observation that asthmatic children seemed to have reduced IgG1 galactosylation levels as well. This indicates that IgG1 galactosylation levels could be used as a biomarker for immune activation of populations, providing a valuable tool for studies examining the epidemiological transition from communicable to non-communicable diseases.
Assuntos
Doenças Transmissíveis/imunologia , Imunoglobulina G/química , Polissacarídeos/química , Receptores Fc/química , Schistosoma/imunologia , Esquistossomose/imunologia , Adolescente , Animais , Biomarcadores/química , Proteína C-Reativa/metabolismo , Criança , Pré-Escolar , Equador , Feminino , Gabão , Alemanha , Gana , Glicosilação , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Inflamação/imunologia , Masculino , Países Baixos , Esquistossomose/parasitologiaRESUMO
C-reactive protein (CRP) is a pattern-recognition molecule, which can bind to phosphorylcholine and certain phosphorylated carbohydrates found on the surface of a number of microorganisms. CRP has been shown recently to bind human Fc receptor for immunoglobulin G (IgG; FcgammaR)I and mediate phagocytosis and signaling through the gamma-chain. To date, binding of monomeric CRP to FcgammaRII has been contentious. We demonstrate that erythrocytes opsonized with CRP bind FcgammaRIIa-transfected COS-7 cells. In addition, we demonstrate that FcgammaRI can use FcgammaRIIa R131 and H131 to phagocytose erythrocytes coated with IgG or purified or recombinant CRP in the absence of the gamma-chain. COS-7 cells expressing FcgammaRIIa or FcgammaRI alone did not phagocytose opsonized erythrocytes. Such phagocytosis required the cytoplasmic domain of FcgammaRIIa, as mutation of tyrosine at position 205 and truncation of the cytoplasmic domain from the end of the transmembrane region (position 206), resulting in the loss of the immunoreceptor tyrosine activatory motif, abrogated phagocytosis. FcgammaRIIa R131 was more efficient than FcgammaRIIa H131 at mediating CRP-dependent phagocytosis.
Assuntos
Antígenos CD/metabolismo , Proteína C-Reativa/metabolismo , Imunoglobulina G/metabolismo , Fagocitose , Receptores de IgG/metabolismo , Animais , Antígenos CD/genética , Células COS , Chlorocebus aethiops , Eritrócitos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Mutação , Receptores de IgG/genética , Proteínas Recombinantes/metabolismo , Ovinos , Transfecção , Tirosina/químicaRESUMO
Complementary (antisense) peptide mini-receptor inhibitors are complementary peptides designed to be receptor-surrogates that act by binding to selected surface features of biologically important proteins thereby inhibiting protein-cognate receptor interactions and subsequent biological effects. Previously, we described a complementary peptide mini-receptor inhibitor of interleukin-1beta (IL-1beta) that was designed to bind to an external surface loop (beta-bulge) of IL-1beta (Boraschi loop) clearly identified in the X-ray crystal structure of this cytokine. Here, we report the de-novo design and rational development of a complementary peptide mini-receptor inhibitor of cytokine interleukin-18 (IL-18), a protein for which there is no known X-ray crystal structure. Using sequence homology comparisons with IL-1beta, putative IL-18 surface loops are identified and used as a starting point for design, including a loop region 1 thought to be equivalent with the Boraschi loop of IL-1beta. Only loop region 1 complementary peptides are found to be promising leads as mini-receptor inhibitors of IL-18 but these are prevented from being properly successful owing to solubility problems. The application of "M-I pair mutagenesis" and inclusion of a C-terminal arginine residue are then sufficient to solve this problem and convert one lead peptide into a functional complementary peptide mini-receptor inhibitor of IL-18. This suggests that the biophysical and biological properties of complementary peptides can be improved in a rational and logical manner where appropriate, further strengthening the potential importance of complementary peptides as inhibitors of protein-protein interactions, even when X-ray crystal structural information is not readily available.
Assuntos
Desenho de Fármacos , Interleucina-18/antagonistas & inibidores , Mimetismo Molecular , Peptídeos/farmacologia , Receptores de Interleucina/química , Humanos , Subunidade alfa de Receptor de Interleucina-18 , Cinética , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica , Receptores de Interleucina-18 , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaAssuntos
Proteína C-Reativa/metabolismo , Glicoesfingolipídeos/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania mexicana/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Humanos , Leishmania donovani/metabolismo , Leishmania mexicana/metabolismo , MorfogêneseRESUMO
Helminths aggravate anemia and malnutrition among school children. We studied this association in a cross-sectional study of 6- to 23-month-old Zanzibari children (N = 2322) and a sub-sample of 690 children matched on age and helminth infection status. Ascaris, hookworm, and Trichuris infections were diagnosed along with recent fever, malaria infection, mid-upper arm circumference (MUAC) and hemoglobin concentration (Hb). Alpha-1-acid glycoprotein (AGP), C-reactive protein (CRP), height, and weight were measured in the sub-sample. Infected children had higher Hb (beta = 5.44 g/L, P < 0.001) and MUAC-for-age Z score (beta = 0.30 Z, P < 0.001) compared with uninfected children after adjusting for covariates. Although helminths were not associated with inflammation, their association with Hb or MUAC-for-age Z score was modified by inflammation. Malaria-infected children were less likely to be infected with helminths (adjusted odds ratios 0.63 [95% confidence interval: 0.49, 0.81]). Non-anemic, better nourished, or non-malaria-infected children may be more exploratory of their environments and therefore increase their exposure to soil-transmitted helminths.
Assuntos
Anemia/complicações , Helmintíase/complicações , Transtornos da Nutrição do Lactente/complicações , Inflamação/complicações , Malária/complicações , Anemia/epidemiologia , Animais , Helmintíase/epidemiologia , Humanos , Lactente , Transtornos da Nutrição do Lactente/epidemiologia , Inflamação/epidemiologia , Malária/epidemiologia , Tanzânia/epidemiologiaRESUMO
Serum amyloid A (SAA) is the major acute-phase protein in man and most mammals. Recently we demonstrated that SAA binds to many Gram-negative bacteria including Escherichia coli and Pseudomonas aeruginosa through outer membrane protein A (OmpA) family members. Therefore we investigated whether SAA altered the response of innate phagocytic cells to bacteria. Both the percentage of neutrophils containing E coli and the number of bacteria per neutrophil were greatly increased by SAA opsonization, equivalent to the increase seen for serum opsonization. In contrast, no change was seen for Streptococcus pneumoniae, a bacteria that did not bind SAA. Neutrophil reactive oxygen intermediate production in response to bacteria was also increased by opsonization with SAA. SAA opsonization also increased phagocytosis of E coli by peripheral blood mononuclear cell-derived macrophages. These macrophages showed strong enhancement of TNF-alpha and IL-10 production in response to SAA-opsonized E coli and P aeruginosa. SAA did not enhance responses in the presence of bacteria to which it did not bind. These effects of SAA occur at normal concentrations consistent with SAA binding properties and a role in innate recognition. SAA therefore represents a novel innate recognition protein for Gram-negative bacteria.
Assuntos
Bactérias Gram-Negativas/imunologia , Neutrófilos/fisiologia , Proteínas Opsonizantes/farmacologia , Proteína Amiloide A Sérica/imunologia , Proteína Amiloide A Sérica/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Microscopia Confocal , Fagocitose , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Valores de Referência , Explosão Respiratória , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/imunologiaRESUMO
Despite their close physical and functional relationships, alveolar macrophages (AMs) and pulmonary dendritic cells (pulDCs) have rarely been examined together in the context of infection. Using a nonlethal, resolving model of pneumonia caused by intranasal injection of Streptococcus pneumoniae, we demonstrate that AMs and pulDCs exhibit distinct characteristics during pulmonary inflammation. Recruitment of AMs and pulDCs occurred with different kinetics, and increased numbers of AMs resulted mainly from the appearance of a distinct subset of CD11b(High) AMs. Increased numbers of CD11b(High) and CD11b(Low) AMs, but not pulDCs, were recoverable from bronchoalveolar lavage fluid. CD11b expression on AMs was significantly increased by granulocyte-macrophage colony-stimulating factor but not by interleukin-10 or pathogen-associated stimuli. Finally, antibody blockade demonstrated that CD11b was critical for the recruitment of AMs, but not pulDCs, into the lung after pneumococcal challenge. These data demonstrate that there are significant differences between AM and pulDC responses to inflammatory pathogenic stimuli in vivo.
Assuntos
Antígeno CD11b/fisiologia , Células Dendríticas/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Pneumocócica/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Antígeno CD11b/biossíntese , Modelos Animais de Doenças , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Streptococcus pneumoniae/imunologiaRESUMO
Tumor necrosis factor (TNF) has been proposed as a major mediator of host resistance in murine models of Streptococcus pneumoniae infection; in humans, anti-TNF therapies have been implicated in increased susceptibility to pneumococcal infection. Here, we use nonlethal (serotype 6B) and lethal (serotype 3) S. pneumoniae, neutralizing monoclonal antibodies to TNF, and TNF gene-deficient mice to reexamine the role played by TNF in antistreptococcal responses. After nonlethal challenge, primary resistance and all examined parameters of the cellular inflammatory response occurred independently of TNF activity. After lethal challenge, TNF deficiency resulted in more-rapid death but did not affect lung inflammation. However, the livers of the TNF gene-deficient mice, but not of the control mice, exhibited extensive signs of systemic disease. TNF, therefore, is dispensable for a complete cellular pulmonary inflammatory response to S. pneumoniae infection but enhances survival from disseminated lethal infection, at least in part by delaying systemic organ damage.
Assuntos
Infecções Pneumocócicas/fisiopatologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Pulmão/microbiologia , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Neutrófilos/fisiologia , Fagócitos/fisiologia , Infecções Pneumocócicas/sangue , Baço/microbiologia , Baço/patologia , Streptococcus pneumoniae , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
Serum amyloid A (SAA) is the major acute phase protein in man and most mammals. We observed SAA binding to a surprisingly large number of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. The binding was found to be high affinity and rapid. Importantly, this binding was not inhibited by high density lipoprotein with which SAA is normally complexed in serum. Binding was also observed when bacteria were offered serum containing SAA. Ligand blots following SDS-PAGE or two-dimensional gels revealed two major ligands of 29 and 35 kDa that bound SAA when probing with radiolabeled SAA or SAA and monoclonal anti-SAA. Following fractionation the ligand was found in the outer membrane fraction of E. coli and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be outer membrane protein A (OmpA). OmpA-deficient E. coli did not bind SAA, and following purification of OmpA the protein retained binding activity. The ligands on other bacteria were likely to be homologues of OmpA because wild type, but not OprF-deficient, P. aeruginosa bound SAA.