RESUMEN
Background/Aims. Multiple sclerosis (MS) is an autoimmune disorder that affects the central nervous system (CNS) primarily hallmarked by neuroinflammation and demyelination. The activation of astrocytes exerts double-edged sword effects, which perform an integral function in demyelination and remyelination. In this research, we examined the therapeutic effects of the Bu Shen Yi Sui capsule (BSYS), a traditional Chinese medicine prescription, in a cuprizone- (CPZ-) triggered demyelination model of MS (CPZ mice). This research intended to evaluate if BSYS might promote remyelination by shifting A1 astrocytes to A2 astrocytes. Methods. The effects of BSYS on astrocyte polarization and the potential mechanisms were explored in vitro and in vivo utilizing real-time quantitative reverse transcription PCR, immunofluorescence, and Western blotting. Histopathology, expression of inflammatory cytokines (IL-10, IL-1ß, and IL-6), growth factors (TGF-ß, BDNF), and motor coordination were assessed to verify the effects of BSYS (3.02 g/kg/d) on CPZ mice. In vitro, A1 astrocytes were induced by TNF-α (30 ng/mL), IL-1α (3 ng/mL), and C1q (400 ng/mL), following which the effect of BSYS-containing serum (concentration of 15%) on the transformation of A1/A2 reactive astrocytes was also evaluated. Results and Conclusions. BSYS treatment improved motor function in CPZ mice as assessed by rotarod tests. Intragastric administration of BSYS considerably lowered the proportion of A1 astrocytes, but the number of A2 astrocytes, MOG+, PLP+, CNPase+, and MBP+ cells was upregulated. Meanwhile, dysregulation of glutathione peroxidase, malondialdehyde, and superoxide dismutase was reversed in CPZ mice after treatment with BSYS. In addition, the lesion area and expression of proinflammatory cytokines were decreased and neuronal protection factors and anti-inflammatory cytokines were increased. In vitro, BSYS-containing serum suppressed the A1 astrocytic markers' expression and elevated the expression levels of A2 markers in primary astrocytes triggered by C1q, TNF-α, and IL-1α. Importantly, the miR-155/SOCS1 signaling pathway was involved in the modulation of the A1/A2 phenotype shift. Overall, this study demonstrated that BSYS has neuroprotective effects in myelin repair by modulating astrocyte polarization via the miR-155/SOCS1 pathway.
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MicroARNs , Esclerosis Múltiple , Animales , Astrocitos/metabolismo , Sistema Nervioso Central , Complemento C1q/metabolismo , Complemento C1q/farmacología , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Vaina de Mielina , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of Iba-1, complement C1q and CD68 in hippocampus of SAMP8 mice, so as to explore its mechanisms underlying improvement of Alzheimer's disease (AD). METHODS: Twenty-four male SAMP8 mice were randomly and equally divided into model and EA groups, and 12 SAMR1 mice were used as the control group. EA (2 Hz, 1.5-2.0 mA) was applied to "Baihui" (GV20), "Dazhui"(GV14) and "Shen-shu"(BL23) for 20 min once daily in the EA group, each course of treatment was 8 days, with an interval of 2 days between two courses, and the mice were treated for 3 courses. Morris water maze test was performed to assess the learning-memory ability of mice. The positive expression levels of Iba-1 and CD68 proteins in the hippocampus CA1 region were detected by immunohistochemistry. The mRNA and protein expression levels of Iba-1,C1q and CD68 in the hippocampus were detected by real-time PCR and Western blot, separately. RESULTS: Compared with the control group, the average escape latency of Morris water maze test was prolonged in the model group (P<0.01), duration of swimming in the original platform quadrant and the number of original platform crossing were significantly shorter and decreased respectively (P<0.01). Compared with the model group, the average escape latency in the EA group was shortened (P<0.05, P<0.01), the duration of swimming in the original platform quadrant and the number of original platform crossing were significantly prolonged and increased (P<0.01). The immunoactivity of Iba-1 and CD68 in hippocampal CA1 region, and mRNA and protein expression levels of hippocampal Iba-1,C1q and CD68 were significantly up-regulated in the model group in contrast to the control group (P<0.01, P<0.05), and obviously down-regulated except the mRNA expression level of hippocampal Iba-1 in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA can improve the learning and memory ability of SAMP8 mice, which may be associated with its effect in inhibiting of complement C1q-dependent microglial phagocytosis in the hippocampus.
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Electroacupuntura , Animales , Complemento C1q/genética , Complemento C1q/metabolismo , Hipocampo/metabolismo , Masculino , Memoria , Ratones , Microglía/metabolismo , Fagocitosis , ARN Mensajero/metabolismoRESUMEN
The classical complement cascade mediates synapse elimination in the visual thalamus during early brain development. However, whether the primary visual cortex also undergoes complement-mediated synapse elimination during early visual system development remains unknown. Here, we examined microglia-mediated synapse elimination in the visual thalamus and the primary visual cortex of early postnatal C1q and SRPX2 knockout mice. In the lateral geniculate nucleus, deletion of C1q caused a persistent decrease in synapse elimination and microglial synapse engulfment, while deletion of SRPX2 caused a transient increase in the same readouts. In the C1q-SRPX2 double knockout mice, the C1q knockout phenotypes were dominant over the SRPX2 knockout phenotypes, a result which is consistent with SRPX2 being an inhibitor of C1q. We found that genetic deletion of either C1q or SRPX2 did not affect synapse elimination or microglial engulfment of synapses in layer 4 of the primary visual cortex in early brain development. Together, these results show that the classical complement pathway regulates microglia-mediated synapse elimination in the visual thalamus but not the visual cortex during early development of the central nervous system.
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Proteínas de la Membrana/metabolismo , Microglía , Proteínas de Neoplasias/metabolismo , Corteza Visual , Animales , Complemento C1q/genética , Complemento C1q/metabolismo , Ratones , Microglía/metabolismo , Sinapsis/metabolismo , Tálamo/metabolismo , Corteza Visual/metabolismoRESUMEN
The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.
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Lesiones Traumáticas del Encéfalo/genética , Activación de Complemento/genética , Lectina de Unión a Manosa de la Vía del Complemento/genética , Lectinas/genética , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Complemento C1/genética , Complemento C1/metabolismo , Complemento C1q/genética , Complemento C1q/metabolismo , Complemento C1r/genética , Complemento C1r/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Hipocampo/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Ratones Endogámicos C57BL , Microglía/metabolismo , Neostriado/metabolismo , Tálamo/metabolismo , Factores de TiempoRESUMEN
The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.
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Complemento C1q/metabolismo , Ácido Edético/química , Prueba de Histocompatibilidad/métodos , Complemento C1q/química , Ditiotreitol/química , Antígenos HLA/inmunología , Humanos , Inmunoglobulina G/química , Unión ProteicaRESUMEN
ANX005 is a humanized immunoglobulin G4 recombinant antibody against C1q that inhibits its function as the initiating molecule of the classical complement cascade. The safety and tolerability of ANX005 are currently being evaluated in a phase I trial in healthy volunteers ( www.clinicaltrials.gov Identifier: NCT03010046). Inhibition of C1q can be applied therapeutically in a broad spectrum of diseases, including acute antibody-mediated autoimmune disease, such as Guillain-Barré syndrome (GBS), and in chronic diseases of the central nervous system involving complement-mediated neurodegeneration, such as Alzheimer's disease (AD). To support the clinical development of ANX005, several studies were conducted to assess the pharmacology, pharmacokinetics, and potential toxicity of ANX005. ANX-M1, the murine precursor of ANX005, functionally inhibits the classical complement cascade both in vitro and in vivo, to protect against disease pathology in mouse models of GBS and AD. Toxicology studies with ANX005, itself, showed that intravenous administration once weekly for 4 weeks was well tolerated in rats and monkeys, with no treatment-related adverse findings. Serum levels of ANX005 in monkeys correlate with a reduction in free C1q levels both in the serum and in the cerebrospinal fluid. In summary, ANX005 has shown proof of concept in in vitro and in vivo nonclinical pharmacology models, with no toxicity in the 4-week repeat-dose studies in rats and monkeys. The no observed adverse effect level was 200 mg/kg/dose, which is 200-fold higher than the first-in-human starting dose of 1 mg/kg in healthy volunteers.
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Anticuerpos Monoclonales Humanizados/toxicidad , Enfermedades Autoinmunes/tratamiento farmacológico , Complemento C1q/inmunología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Enfermedades Autoinmunes/inmunología , Complemento C1q/metabolismo , Vía Clásica del Complemento/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Enfermedades Neurodegenerativas/inmunología , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment.
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Complejo Antígeno-Anticuerpo/inmunología , Artritis Reumatoide/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Polisacáridos/química , Polisacáridos/inmunología , Adulto , Anciano , Complejo Antígeno-Anticuerpo/química , Artritis Reumatoide/terapia , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C3c/inmunología , Complemento C3c/metabolismo , Femenino , Fucosa/metabolismo , Galactosa/metabolismo , Glicosilación , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Lectinas/metabolismo , Masculino , Persona de Mediana Edad , Polisacáridos/metabolismo , Sambucus nigra , Ácidos Siálicos/metabolismoRESUMEN
Chagas disease is an endemic pathology in Latin America, now emerging in developed countries, caused by the intracellular protozoan Trypanosoma cruzi, whose life cycle involves three stages: amastigotes, epimastigotes, and trypomastigotes. T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum resident chaperone, translocates to the external cellular membrane, where it captures complement component C1, ficolins and MBL, thus inactivating the classical and lectin pathways. Trypomastigote-bound C1 is detected as an "eat me" signal by macrophages and promotes the infective process. Unlike infective trypomastigotes, non-infective epimastigotes either do not express or express only marginal levels of TcCRT on their external membrane. We show that epimastigotes bind exogenous rTcCRT to their cellular membrane and, in the presence of C1q, this parasite form is internalized into normal fibroblasts. On the other hand, Calreticulin (CRT)-deficient fibroblasts show impaired parasite internalization. In synthesis, CRT from both parasite and host cell origin is important in the establishment of C1q-dependent first contacts between parasites and host cells.
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Calreticulina/inmunología , Endocitosis/inmunología , Interacciones Huésped-Parásitos/inmunología , Trypanosoma cruzi/inmunología , Adyuvantes Inmunológicos , Animales , Calreticulina/genética , Calreticulina/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Fibroblastos/metabolismo , Fibroblastos/parasitología , Técnicas de Inactivación de Genes , Ratones , Unión Proteica , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidad , Factores de Virulencia/inmunologíaRESUMEN
The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.
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Complemento C1q/metabolismo , Células Dendríticas/inmunología , Eosinófilos/inmunología , Hipersensibilidad/inmunología , Pulmón/inmunología , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Alérgenos/inmunología , Animales , Betula , Células Cultivadas , Complemento C1q/administración & dosificación , Femenino , Humanos , Interleucina-10/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Extractos Vegetales , Polen/inmunologíaRESUMEN
Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.
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Envejecimiento/metabolismo , Encéfalo/metabolismo , Activación de Complemento , Complemento C1q/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microglía/metabolismo , Envejecimiento/inmunología , Animales , Líquido Cefalorraquídeo , Complemento C1q/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Granulinas , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Lisosomas/metabolismo , Redes y Vías Metabólicas , Ratones , Trastorno Obsesivo Compulsivo/genética , Trastorno Obsesivo Compulsivo/metabolismo , Progranulinas , Sinapsis/metabolismo , Tálamo/metabolismoRESUMEN
BACKGROUND: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR). MATERIAL AND METHODS: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS. RESULTS: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process. CONCLUSIONS: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.
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Calreticulina/metabolismo , Sistema Nervioso Central/lesiones , Sistema Nervioso Central/patología , Complemento C1q/metabolismo , Hirudo medicinalis/metabolismo , Microglía/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biotinilación , Calreticulina/química , Calreticulina/genética , Sistema Nervioso Central/metabolismo , Quimiotaxis , Humanos , Microglía/patología , Datos de Secuencia Molecular , Filogenia , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , SolubilidadRESUMEN
OBJECTIVES: To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. METHODS: Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. RESULTS: Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. CONCLUSIONS: In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.
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Transfusión de Sangre Autóloga , Procedimientos Quirúrgicos Cardíacos , Puente Cardiopulmonar , Micropartículas Derivadas de Células/fisiología , Activación de Complemento/fisiología , Pericardio/fisiopatología , Proteína C-Reactiva/metabolismo , Complemento C1q/metabolismo , Citometría de Flujo , Humanos , Inmunoglobulina M/metabolismo , Componente Amiloide P Sérico/metabolismoRESUMEN
Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated TH2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-γ (IFNγ) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFNγ, in the spinal cord implicating IFNγ in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFNγ or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the TH1/TH2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing TH1 immunity. Thus, because of its lower TH1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.
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Esclerosis Amiotrófica Lateral/terapia , Superóxido Dismutasa/genética , Superóxido Dismutasa/inmunología , Vacunación/métodos , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/patología , Análisis de Varianza , Animales , Cromatografía en Gel/métodos , Complemento C1q/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Regulación de la Expresión Génica/inmunología , Humanos , Inmunidad/genética , Inmunidad/fisiología , Inmunoglobulina G , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Mutación , Transducción de Señal/genética , Médula Espinal/inmunología , Médula Espinal/metabolismo , Médula Espinal/patología , Bazo/metabolismo , Superóxido Dismutasa-1 , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoAsunto(s)
Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Activación de Complemento/efectos de los fármacos , Depleción Linfocítica/métodos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales de Origen Murino , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Antígenos CD20/metabolismo , Antineoplásicos/inmunología , Antineoplásicos/metabolismo , Activación de Complemento/genética , Activación de Complemento/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Mutación Missense , Unión Proteica/genética , Unión Proteica/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunología , RituximabRESUMEN
Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG(2)a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Activación de Complemento/efectos de los fármacos , Depleción Linfocítica/métodos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales de Origen Murino , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Antígenos CD20/metabolismo , Antineoplásicos/inmunología , Antineoplásicos/metabolismo , Activación de Complemento/genética , Activación de Complemento/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Mutación Missense , Unión Proteica/genética , Unión Proteica/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunología , RituximabRESUMEN
The deposition of amyloid beta-protein (A beta) in cerebral vasculature, known as cerebral amyloid angiopathy (CAA), is a common pathological feature of Alzheimer's disease and related disorders. In familial forms of CAA single mutations in the A beta peptide have been linked to the increase of vascular A beta deposits accompanied by a strong localized activation of glial cells and elevated expression of neuroinflammatory mediators including complement proteins. We have developed human amyloid-beta precursor protein transgenic mice harboring two CAA A beta mutations (Dutch E693Q and Iowa D694N) that mimic the prevalent cerebral microvascular A beta deposition observed in those patients, and the Swedish mutations (K670N/M671L) to increase A beta production. In these Tg-SwDI mice, we have reported predominant fibrillar A beta along microvessels in the thalamic region and diffuse plaques in cortical region. Concurrently, activated microglia and reactive astrocytes have been detected primarily in association with fibrillar cerebral microvascular A beta in this model. Here we show that three native complement components in classical and alternative complement pathways, C1q, C3, and C4, are elevated in Tg-SwDI mice in regions rich in fibrillar microvascular A beta. Immunohistochemical staining of all three proteins was increased in thalamus, hippocampus, and subiculum, but not frontal cortex. Western blot analysis showed significant increases of all three proteins in the thalamic region (with hippocampus) as well as the cortical region, except C3 that was below detection level in cortex. Also, in the thalamic region (with hippocampus), C1q and C3 mRNAs were significantly up-regulated. These complement proteins appeared to be expressed largely by activated microglial cells associated with the fibrillar microvascular A beta deposits. Our findings demonstrate that Tg-SwDI mice exhibit elevated complement protein expression in response to fibrillar vascular A beta deposition that is observed in patients with familial CAA.
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Péptidos beta-Amiloides/genética , Angiopatía Amiloide Cerebral/metabolismo , Complemento C1q/metabolismo , Complemento C3/metabolismo , Complemento C4/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/irrigación sanguínea , Angiopatía Amiloide Cerebral/genética , Angiopatía Amiloide Cerebral/patología , Complemento C1q/genética , Complemento C3/genética , Complemento C4/genética , Modelos Animales de Enfermedad , Hipocampo/irrigación sanguínea , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Mutación/genética , Tálamo/irrigación sanguínea , Tálamo/metabolismo , Tálamo/patologíaRESUMEN
Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.
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Anticuerpos Monoclonales/administración & dosificación , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Colágeno Tipo II/inmunología , Vía Alternativa del Complemento/inmunología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Complejo Antígeno-Anticuerpo/fisiología , Artritis Experimental/genética , Calcio/deficiencia , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Complemento C1q/deficiencia , Complemento C1q/genética , Complemento C1q/metabolismo , Complemento C3/deficiencia , Complemento C3/genética , Complemento C3/metabolismo , Vía Alternativa del Complemento/genética , Femenino , Sueros Inmunes/genética , Sueros Inmunes/fisiología , Masculino , Lectina de Unión a Manosa/sangre , Lectina de Unión a Manosa/deficiencia , Lectina de Unión a Manosa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/genética , Unión Proteica/inmunología , Zimosan/farmacologíaRESUMEN
In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.
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Artritis/sangre , Glucosa-6-Fosfato Isomerasa/sangre , Inflamación/metabolismo , Articulaciones/patología , Líquido Sinovial/metabolismo , Artritis/metabolismo , Estudios de Cohortes , Complemento C1q/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosa-6-Fosfato Isomerasa/metabolismo , Humanos , Sistema Inmunológico , Masculino , Proteínas Recombinantes/químicaRESUMEN
The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.
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Arginina , Complemento C1q/genética , Histidina , Inmunoglobulina G/metabolismo , Subunidades de Proteína/genética , Proteínas Recombinantes/genética , Alanina/genética , Animales , Arginina/genética , Proteínas Inactivadoras del Complemento 1/fisiología , Complemento C1q/antagonistas & inhibidores , Complemento C1q/metabolismo , Análisis Mutacional de ADN/métodos , Eritrocitos/inmunología , Escherichia coli/genética , Hemólisis/inmunología , Histidina/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación Puntual , Subunidades de Proteína/fisiología , OvinosRESUMEN
Allergen extracts are efficient activators of the complement system trough the classical pathway. Involvement of the lectin pathway was not previously studied. To further examine the mechanism of complement activation by allergens, in vitro experiments, which covered early steps both of classical and lectin pathways, were performed. Two types of allergens used in these studies: parietaria (PA) and house dust (HD) mite extracts. These allergen extracts bound to the globular head of C1q and interacted with purified mannan-binding lectin (MBL) as measured by solid-phase ELISA. None of the allergen extracts was able to activate human C1 in vitro, as measured by the determination of the split products of C1s in a reconstituted precursor C1 preparation. Neither the HD nor the PA extracts induced C4d generation above background in the serum of three subjects with hypogammaglobulinaemia but normal complement haemolytic activity. After reconstitution to normal level with purified human IgG, allergen extracts induced C4d formation above control at a level comparable to that measured in normal serum incubated with the same amounts of the extracts. HD-induced C4d generation was about the same comparable in MBL-depleted serum and in normal sera. In contrast PA induced no C4d formation in the MBL-depleted serum, whereas reconstitution with purified MBL restored C4d generation. These in vitro findings indicate that although the allergen extracts can bind purified C1q and MBL, they require IgG for efficient complement activation. Depending on the allergens, this activation may be initiated through C1, MBL, or both.