RESUMEN
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.
Asunto(s)
Proteína C-Reactiva/inmunología , Inmunidad Innata , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/inmunología , Animales , Proteína C-Reactiva/deficiencia , Proteína C-Reactiva/genética , Humanos , Ratones , Ratones Noqueados , FenotipoRESUMEN
Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.
Asunto(s)
Amiloide/metabolismo , Amiloidosis/fisiopatología , Inflamación/complicaciones , Proteína Amiloide A Sérica/metabolismo , Amiloidosis/etiología , Animales , Rojo Congo , Cartilla de ADN/genética , Doxiciclina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.
Asunto(s)
Proteína C-Reactiva/análisis , Citocinas/metabolismo , Leucocitos Mononucleares/metabolismo , Componente Amiloide P Sérico/análisis , Proteínas de Fase Aguda/análisis , Proteínas de Fase Aguda/aislamiento & purificación , Proteínas de Fase Aguda/farmacología , Amiloidosis/sangre , Animales , Proteína C-Reactiva/aislamiento & purificación , Proteína C-Reactiva/farmacología , Células Cultivadas , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Componente Amiloide P Sérico/aislamiento & purificación , Componente Amiloide P Sérico/farmacología , Espectrometría de Masa por Ionización de Electrospray , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Human serum amyloid P component (SAP) is of increasing interest for its possible pathogenic role in amyloidosis and Alzheimer's disease, and as a therapeutic target in these conditions. We have developed and validated a robust and reproducible immunoradiometric assay (IRMA) for human SAP in serum, plasma and cerebrospinal fluid, and characterized the notable stability of human SAP immunoreactivity during storage of undiluted serum at 4°C and 37°C as well as frozen at -30°C. SAP values were also stable after repeated freeze thawing of highly diluted serum samples. The 100 fold dynamic range of the assay, 0.5-50 µg/L, encompassed all values seen in blood and cerebrospinal fluid, when tested at suitable dilutions, from both normal healthy individuals and patients, including subjects receiving the SAP-depleting drug, CPHPC. Furthermore by comparing the IRMA values in the presence and absence of calcium, the new assay revealed interference due to the binding of CPHPC by SAP, which was markedly enhanced in heparinized plasma. It is therefore essential that SAP assays in samples from patients on CPHPC be conducted in the absence of free calcium, in order to completely abrogate interference and determine the actual total SAP concentration. Estimates by the IRMA of SAP concentration in 49 serum samples from amyloidosis patients corresponded closely with those obtained by the established standard electro-immunoassay method and by a newly developed commercial ELISA kit (Hycult Biotechnology).
Asunto(s)
Ensayo Inmunorradiométrico/métodos , Componente Amiloide P Sérico/análisis , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/tratamiento farmacológico , Amiloidosis/sangre , Amiloidosis/tratamiento farmacológico , Análisis Químico de la Sangre/métodos , Ácidos Carboxílicos/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoensayo , Estabilidad Proteica , Pirrolidinas/farmacología , Componente Amiloide P Sérico/antagonistas & inhibidoresRESUMEN
Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.
Asunto(s)
Amiloide/efectos de los fármacos , Amiloidosis/prevención & control , Anticuerpos/inmunología , Anticuerpos/farmacología , Componente Amiloide P Sérico/antagonistas & inhibidores , Componente Amiloide P Sérico/inmunología , Amiloidosis/terapia , Animales , Anticuerpos/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Componente Amiloide P Sérico/genéticaRESUMEN
Serum amyloid P component (SAP) is a universal constituent of amyloid deposits and contributes to their formation and/or persistence. We therefore developed CPHPC ((R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexa-noyl]pyrrolidine-2 carboxylic acid), a novel bis(D-proline) drug, to specifically target SAP and report here a first, exploratory, open label proof of principle study in systemic amyloidosis. CPHPC produced sustained, >95% depletion of circulating SAP in all patients and c. 90% reduction in the SAP content of the two amyloidotic organs that became available. There were no significant adverse effects of either SAP depletion or CPHPC itself. No accumulation of amyloid was demonstrable by SAP scintigraphy in any patient on the drug. In hereditary fibrinogen amyloidosis, which is inexorably progressive, proteinuria was reduced in four of five patients receiving CPHPC and renal survival was prolonged compared to a historical control group. These promising clinical observations merit further study.
Asunto(s)
Amiloidosis/tratamiento farmacológico , Amiloidosis/metabolismo , Ácidos Carboxílicos/uso terapéutico , Componente Amiloide P Sérico/efectos de los fármacos , Componente Amiloide P Sérico/metabolismo , Adulto , Anciano , Amiloidosis/sangre , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteinuria/tratamiento farmacológicoRESUMEN
New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Ácidos Carboxílicos/administración & dosificación , Pirrolidinas/administración & dosificación , Componente Amiloide P Sérico/antagonistas & inhibidores , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Espectrometría de Masas , Persona de Mediana Edad , Proyectos Piloto , Conformación Proteica , Componente Amiloide P Sérico/líquido cefalorraquídeo , Componente Amiloide P Sérico/químicaRESUMEN
The pathogenic significance, if any, of the epidemiological association between baseline C-reactive protein (CRP) values and future atherothrombotic events is not known. We therefore investigated spontaneous atherosclerosis and atherothrombosis, and systemic markers of inflammation (acute phase proteins), in aged, normal diet-fed, male apolipoprotein E deficient (apoE(-/-)) mice with and without transgenic expression of human CRP. At 18 months of age, aortic atherosclerosis was extensive but with no significant difference in plaque size between C57BL/6apoE(-/-) mice with (apoE(-/-)-hCRP(+)) and without transgenic human CRP (apoE(-/-)). Atherosclerotic lesions in brachiocephalic arteries were typically complex and layered, with extensive fibrotic-cholesterol deposits, calcification and occasional recent intraplaque haemorrhage and thrombus, but with no significant overall differences between apoE(-/-) and apoE(-/-)-hCRP(+) animals. Concentrations of mouse serum amyloid P component (SAP) were essentially normal throughout and did not differ between apoE(-/-) and apoE(-/-)-hCRP(+) mice, or between wild-type (apoE(+/+)) and apoE(-/-) mice, regardless of human CRP expression. Mouse serum amyloid A protein (SAA), and human CRP concentrations were modestly but significantly higher in apoE(-/-)-hCRP(+) than in apoE(+/+)-hCRP(+) animals, but mouse SAA values were unaffected by transgenic expression of human CRP in either background. Thus, there was no evidence in this 18 month study of apoE(-/-), and control apoE(+/+) mice, that transgenic human CRP was pro-atherogenic, pro-inflammatory or pro-atherothrombotic.
Asunto(s)
Aterosclerosis/fisiopatología , Proteína C-Reactiva/fisiología , Trombosis/fisiopatología , Envejecimiento , Animales , Apolipoproteínas E/genética , Proteína C-Reactiva/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones TransgénicosAsunto(s)
Proteína C-Reactiva/metabolismo , Leptina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Proteína C-Reactiva/farmacología , Ingestión de Alimentos/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Leptina/sangre , Leptina/farmacología , Ratones , Unión ProteicaRESUMEN
The association between circulating concentrations of C-reactive protein (CRP) and future atherothrombotic events has provoked speculation about a possible pathogenetic role of CRP. However, we show here that transgenic expression of human CRP had no effect on development, progression, or severity of spontaneous atherosclerosis, or on morbidity or mortality, in male apolipoprotein E (apoE)-deficient C57BL/6 mice up to 56 weeks, despite deposition of human CRP and mouse complement component 3 in the plaques. Although female apoE knockouts develop atherosclerosis more rapidly than males, the human CRP transgene is under sex hormone control and is expressed at human levels only in males. We therefore studied only male mice. The concentration of mouse serum amyloid P component, an extremely sensitive systemic marker of inflammation, remained normal throughout except for transient spikes in response to fighting in a few animals, indicating that atherogenesis in this model is not associated with an acute-phase response. However, among human CRP transgenic mice, the circulating CRP concentration was higher in apoE knockouts than in wild-type controls. The higher CRP values were associated with substantially lower estradiol concentrations in the apoE-deficient animals. Human CRP transgene expression is thus up-regulated in apoE-deficient mice, apparently reflecting altered estrogen levels, despite the absence of other systemic signs of inflammation. Extrapolation to human pathology from this xenogeneic combination of human CRP with apoE deficiency-mediated mouse atherosclerosis must be guarded. Nevertheless, the present results do not suggest that human CRP is either proatherogenic or atheroprotective in vivo.
Asunto(s)
Apolipoproteínas E/deficiencia , Arteriosclerosis/genética , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Transgenes/genética , Reacción de Fase Aguda , Envejecimiento/sangre , Animales , Antígenos de Diferenciación/metabolismo , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Complemento C3/metabolismo , Estradiol/sangre , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Testosterona/sangre , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
During induction of reactive systemic amyloid A protein (AA) amyloidosis in mice, either by chronic inflammation or by severe acute inflammation following injection of amyloid enhancing factor, the earliest deposits form in a perifollicular distribution in the spleen. Because the splenic follicular localization of immune complexes and of the scrapie agent are both complement dependent in mice, we investigated the possible complement dependence of AA amyloid deposition. In preliminary experiments, substantial depletion of circulating C3 by cobra venom factor had little effect on experimental amyloid deposition. More importantly, mice with targeted deletion of the genes for C1q or for both factor B and C2, and therefore unable to sustain activation, respectively, of either the classical complement pathway or both the classical and alternative pathways, showed amyloid deposition similar to wild type controls. Complement activation by either the classical or alternative pathways is thus not apparently necessary for the experimental induction of systemic AA amyloid in mice.
Asunto(s)
Amiloidosis/inmunología , Vía Alternativa del Complemento , Vía Clásica del Complemento , Proteína Amiloide A Sérica/metabolismo , Amiloidosis/inducido químicamente , Amiloidosis/metabolismo , Animales , Complemento C1q/genética , Complemento C1q/inmunología , Complemento C2/genética , Complemento C2/inmunología , Complemento C3/inmunología , Factor B del Complemento/genética , Factor B del Complemento/inmunología , Venenos Elapídicos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones NoqueadosRESUMEN
Human serum amyloid P component (SAP) binds avidly to DNA, chromatin and apoptotic cells in vitro and in vivo. 129/Sv x C57BL/6 mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoantibodies and immune complex glomerulonephritis. SAP-deficient animals, created by backcrossing the 129/Sv SAP gene deletion into pure line C57BL/6 mice and studied here for the first time, also spontaneously developed broad spectrum antinuclear autoimmunity and proliferative immune complex glomerulonephritis but without proteinuria, renal failure, or increased morbidity or mortality. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP(-/-) mice than in wild-type mice. In contrast, SAP-deficient pure line 129/Sv mice did not produce significant autoantibodies either spontaneously or when immunized with extrinsic chromatin or apoptotic cells, indicating that loss of tolerance is markedly strain dependent. However, SAP deficiency in C57BL/6 mice only marginally affected plasma clearance of exogenous chromatin and had no effect on distribution of exogenous nucleosomes between the liver and kidneys, which were the only tissue sites of catabolism. Furthermore, transgenic expression of human SAP in the C57BL/6 SAP knockout mice did not abrogate the autoimmune phenotype. This may reflect the different binding affinities of mouse and human SAP for nuclear autoantigens and/or the heterologous nature of transgenic human SAP in the mouse. Alternatively, the autoimmunity may be independent of SAP deficiency and caused by expression of 129/Sv chromosome 1 genes in the C57BL/6 background.
Asunto(s)
Autoinmunidad/inmunología , Glomerulonefritis/inmunología , Enfermedades del Complejo Inmune/inmunología , Componente Amiloide P Sérico/genética , Animales , Anticuerpos Antinucleares/biosíntesis , Apoptosis/inmunología , Cromatina/inmunología , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Humanos , Enfermedades del Complejo Inmune/patología , Enfermedades del Complejo Inmune/fisiopatología , Inmunización , Riñón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nucleosomas/metabolismo , Factor Reumatoide/biosíntesis , Componente Amiloide P Sérico/deficienciaRESUMEN
The authors sought to assess the repeatability of measurements of C-reactive protein, an independent predictor of coronary heart disease, in a large cohort of apparently healthy men and to correct earlier estimates of the association of C-reactive protein and coronary heart disease for the measurement error in this protein. They measured C-reactive protein by a high-sensitivity assay in 936 men aged 45-64 years in the MONICA (Monitoring of Trends and Determinants in Cardiovascular Disease) Augsburg cohort in 1984-1985 and remeasured it 3 years later. All men were subjected to an 8-year follow-up of their cardiovascular status. The analytical variation of the assay was small, with the analytical variance component at 1 percent of the within-subject variance component, a repeatability coefficient of 25 percent, and a reliability coefficient of 1.00. In contrast, the within-subject variation of C-reactive protein corresponded to a repeatability coefficient of 740 percent and a reliability coefficient of 0.54, indicating considerable within-subject variation. Based on the authors' estimates, three serial determinations of C-reactive protein should be done to achieve a reliability of 0.75, the value they found for total cholesterol. Correcting the hazard ratios in their original analysis of the association of coronary heart disease and high-sensitivity-assay C-reactive protein for the measurement error in C-reactive protein and covariables leads to a considerably larger estimate. The results suggest that the true association between C-reactive protein and cardiovascular risk is underestimated by a single C-reactive protein determination, and that several serial C-reactive protein measurements should be taken.
Asunto(s)
Biomarcadores/sangre , Proteína C-Reactiva/análisis , Enfermedad Coronaria/sangre , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Binding of serum amyloid P component (SAP) to its ligands, including bacteria, chromatin and amyloid fibrils, protects them from degradation, is anti-opsonic and anti-immunogenic. SAP thereby enhances the virulence of pathogenic bacteria to which it binds. However SAP also contributes to host resistance against bacteria to which it does not bind. Human SAP has been reported to bind to the influenza virus and inhibit viral invasion of cells in tissue culture. We therefore investigated a possible role of SAP in either host resistance or viral virulence during influenza infection in vivo. MATERIALS AND METHODS: The clinical course of mouse adapted influenza virus infection, the host antibody response, and viral replication, were compared in wild type mice, mice with targeted deletion of the SAP gene, and mice transgenic for human SAP. The effects of reconstitution of SAP deficient mice with pure human SAP, and of a drug that specifically blocks SAP binding in vivo, were also studied. Binding of mouse and human SAP to immobilized influenza virus was compared. RESULTS: The presence, absence, or availability for binding of SAP in vivo had no significant or consistent effect on the course or outcome of influenza infection, or on either viral replication or the anti-viral antibody response. Mouse SAP bound much less avidly than human SAP to influenza virus. CONCLUSIONS: In marked contrast to the dramatic effects of SAP deficiency on host resistance to different bacterial infections, mouse SAP apparently plays no significant role during infection of mice with influenza virus. Human SAP binds much more avidly than mouse SAP to the virus, but also had no effect on any of the parameters measured and is therefore unlikely to be involved in human influenza infection.