RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1. METHODS: We developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation. RESULTS: The ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001). CONCLUSIONS: These results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Adulto , Humanos , Camundongos , Animais , Glipicanas/metabolismo , Glipicanas/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Imunoterapia , Epitopos , Imunoglobulina M , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
ß2-glycoprotein I (ß2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). ß2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound ß2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through ß2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target ß2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound ß2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.
Assuntos
Síndrome Antifosfolipídica , Tromboembolia , Trombose , Animais , Camundongos , Ratos , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , beta 2-Glicoproteína I , Células Endoteliais , Trombose/tratamento farmacológico , Trombose/etiologiaRESUMO
ß2-Glycoprotein I (ß2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to ß2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of ß2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and ß2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to ß2-GPI was detected on the surface of HUVECs, and colocalization of MBL with ß2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because ß2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the ß2-GPI/MBL complex may contribute to amplify similar activities of anti-ß2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.
Assuntos
Ativação do Complemento/imunologia , Lectina de Ligação a Manose/metabolismo , Trombina/metabolismo , beta 2-Glicoproteína I/metabolismo , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Cálcio/metabolismo , Linhagem Celular , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio/imunologia , Endotélio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lectina de Ligação a Manose/imunologia , Ligação Proteica/imunologia , Trombina/imunologia , Trombose/imunologia , Trombose/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , beta 2-Glicoproteína I/imunologiaRESUMO
Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34â¯+â¯endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34â¯+â¯endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34â¯+â¯endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation.
Assuntos
Artrite Reumatoide/terapia , Células Endoteliais/imunologia , Inflamação/terapia , Metotrexato/uso terapêutico , Nanopartículas/uso terapêutico , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Animais , Antígenos CD34/metabolismo , Modelos Animais de Doenças , Humanos , Nanopartículas/química , Neovascularização Patológica , Poliésteres/química , Ratos , Ratos WistarRESUMO
Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of ß2glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of ß2glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-ß2glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound ß2glycoproteinI while being able to interact with fluid-phase ß2glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo, and the interaction of these antibodies with circulating ß2glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining "really at risk" patients for more appropriate treatments to avoid recurrences and disability.
Assuntos
Síndrome Antifosfolipídica , Autoanticorpos , Imunoglobulina G , Isquemia Mesentérica , beta 2-Glicoproteína I , Animais , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Complemento C3/imunologia , Complemento C3/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Isquemia Mesentérica/sangue , Isquemia Mesentérica/induzido quimicamente , Isquemia Mesentérica/imunologia , Domínios Proteicos , Ratos , Ratos Wistar , beta 2-Glicoproteína I/sangue , beta 2-Glicoproteína I/imunologiaRESUMO
The abortion-prone mating combination CBA/J × DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J × DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J × DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss.
Assuntos
Lectina de Ligação a Manose da Via do Complemento , Pré-Eclâmpsia/tratamento farmacológico , Animais , Anticorpos Bloqueadores/administração & dosagem , Complemento C5/imunologia , Complemento C5/metabolismo , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Implantação do Embrião/efeitos dos fármacos , Feminino , Humanos , Masculino , Lectina de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Pré-Eclâmpsia/imunologia , GravidezRESUMO
A single-chain fragment variable (scFv) recognizing ß2-glycoprotein 1 (ß2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against ß2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-ß2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-ß2GPI antibodies from APS patients and displaced ß2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/imunologia , Autoantígenos/imunologia , Proteínas do Sistema Complemento/imunologia , beta 2-Glicoproteína I/imunologia , Aborto Espontâneo/imunologia , Animais , Anticorpos Monoclonais/genética , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos , Ligação Proteica/imunologia , Ratos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/uso terapêutico , Trombose/imunologia , Trofoblastos , beta 2-Glicoproteína I/metabolismoRESUMO
OBJECTIVE: Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response. METHODS: Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell-attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system. RESULTS: NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA. CONCLUSION: We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ.
Assuntos
Imunidade Adaptativa/imunologia , Artrite Infecciosa/imunologia , Proteínas de Bactérias/imunologia , Quimiocinas CXC/imunologia , Doença de Lyme/imunologia , Animais , Artrite Infecciosa/etiologia , Artrite Infecciosa/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Quimiocinas/análise , Quimiocinas/metabolismo , Quimiocinas CXC/administração & dosagem , Quimiocinas CXC/metabolismo , Quimiotaxia/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Injeções Intra-Articulares , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Doença de Lyme/complicações , Doença de Lyme/patologia , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Ratos , Joelho de Quadrúpedes/efeitos dos fármacos , Joelho de Quadrúpedes/patologia , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Membrana Sinovial/patologia , Linfócitos T/metabolismo , Linfócitos T/patologiaAssuntos
Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/imunologia , Ativação do Complemento/imunologia , Trombectomia/efeitos adversos , Trombose/etiologia , Anticorpos Antifosfolipídeos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Síndrome Antifosfolipídica/sangue , Feminino , Artéria Femoral/patologia , Artéria Femoral/cirurgia , Humanos , Pessoa de Meia-Idade , Trombose/prevenção & controle , Trombose/cirurgiaRESUMO
In vitro studies have documented ß2 glycoprotein I (ß2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of ß2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled ß2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The ß2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar ß2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after ß2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that ß2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Trofoblastos/metabolismo , Útero/metabolismo , beta 2-Glicoproteína I/sangue , Animais , Complemento C1q/metabolismo , Complemento C3/metabolismo , Complemento C9/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Morte Fetal/sangue , Morte Fetal/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Trofoblastos/patologia , Útero/irrigação sanguínea , Útero/patologiaRESUMO
OBJECTIVE: To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. METHODS: Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody-induced arthritis (CAIA). RESULTS: MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. CONCLUSION: Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Complemento C5/imunologia , Membrana Sinovial/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Colágeno/efeitos adversos , Modelos Animais de Doenças , Endotélio/metabolismo , Adjuvante de Freund/efeitos adversos , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Proteínas Recombinantes/uso terapêutico , Soroalbumina Bovina/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at tissue level. The mechanisms and pathways of local complement activation remain unclear. METHODS: We performed immunofluorescence analyses of autopsy specimens of lungs, kidney and liver from nine COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG and spike protein of SARS-CoV-2. FINDINGS: Lung deposits of C1q, C4, C3 and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. Factor B deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on hepatic artery and portal vein of the liver. INTERPRETATION: Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease. FUNDING: Grants from the Italian Ministry of Health (COVID-2020-12371808) to PLM and National Institutes of Health HL150146 to NP are gratefully acknowledged.
RESUMO
Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at the tissue level. The mechanisms and pathways of local complement activation remain unclear. The aim of this study was to investigate the deposition of complement components in the lungs, kidneys, and liver in patients with COVID-19 patients and to determine the pathway/s of complement activation. We performed immunofluorescence analyses of autopsy specimens of lungs, kidney, and liver from 12 COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG, and spike protein of SARS-CoV-2. Lung deposits of C1q, C4, C3, and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. FB deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on the hepatic artery and portal vein of the liver. Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease and the contribution of the complement system to inflammation and tissue damage.
RESUMO
BACKGROUND: Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. OBJECTIVE: Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. METHODS: The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. RESULTS: We observed that the attack phase plasma from C1 inhibitor-deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor-high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1beta and was markedly reduced by brefeldin A. CONCLUSION: Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.
Assuntos
Angioedema/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Proteína Inibidora do Complemento C1/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptores de Complemento/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Angioedema/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/imunologia , Vasos Sanguíneos/metabolismo , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Bradicinina/uso terapêutico , Antagonistas de Receptor B1 da Bradicinina , Brefeldina A/farmacologia , Permeabilidade Capilar/fisiologia , Linhagem Celular Tumoral , Proteína Inibidora do Complemento C1/genética , Angioedema Hereditário Tipos I e II/tratamento farmacológico , Angioedema Hereditário Tipos I e II/fisiopatologia , Humanos , Fatores Imunológicos/farmacologia , Interleucina-1beta/farmacologia , Masculino , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Endogâmicos WKYRESUMO
Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, ß2 glycoprotein I (ß2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.
Assuntos
Aborto Habitual/etiologia , Síndrome Antifosfolipídica/complicações , Trombose/etiologia , Aborto Habitual/imunologia , Síndrome Antifosfolipídica/imunologia , Feminino , Humanos , Gravidez , Recidiva , Trombose/imunologia , beta 2-Glicoproteína I/metabolismoRESUMO
Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney.
RESUMO
Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa(-/-)) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa(-/-) mice. Bone marrow (BM) chimeras between C1qa(-/-) and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.
Assuntos
Ativação do Complemento/fisiologia , Complemento C1q/metabolismo , Neoplasias/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Complemento C1q/genética , Complemento C3/genética , Complemento C3/metabolismo , Complemento C5/genética , Complemento C5/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production.
Assuntos
Anticorpos Neutralizantes/imunologia , Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Complemento C5/imunologia , Animais , Artrite Experimental/genética , Linhagem Celular , Ativação do Complemento/imunologia , Complemento C5/genética , Cricetinae , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/prevenção & controle , Injeções Intra-Articulares , Masculino , Ratos , Ovinos , TransfecçãoRESUMO
BACKGROUND: Complement activation has been implicated in the development of posttransplant ischemia-reperfusion (I/R) which is responsible for the delayed function of 20% to 30% of grafts. C5a and the terminal complement complex (TCC) are the complement activation products mainly involved in tissue injury caused by I/R. METHODS: To control activation of the terminal step of the complement activation pathways, we used a neutralizing minibody to C5 containing a human single-chain fragment variable (scFv) linked to the hinge region, CH2, and CH3 domains of rat IgG1. RESULTS: The minibody acts on C5 inhibiting the release of C5a and the assembly of TCC and depletes circulating C5 in Sprague-Dawley rats with a therapeutic activity of 4 hr. Administration of the minibody to rats 30 min before heart allotransplantation prevented tissue deposition of TCC, apoptosis, and necrosis of the graft and increase in the levels of serum creatine phosphokinase and tumor necrosis factor-alpha observed in control transplanted rats. CONCLUSIONS: These data suggest that an anti-C5 therapy is effective in preventing graft injury caused by I/R. A minibody containing the human scFv linked to the hinge region and the CH2 and CH3 domains of human IgG1 is ready for use in clinical transplantation.
Assuntos
Complemento C5/uso terapêutico , Transplante de Coração/efeitos adversos , Complicações Pós-Operatórias/prevenção & controle , Traumatismo por Reperfusão/prevenção & controle , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Anticorpos/uso terapêutico , Aorta Abdominal/cirurgia , Aorta Torácica/cirurgia , Ativação do Complemento/efeitos dos fármacos , Complemento C5/imunologia , Transplante de Coração/patologia , Marcação In Situ das Extremidades Cortadas , Injeções Intravenosas , Masculino , Contração Miocárdica , Necrose , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/fisiopatologia , Ratos , Ratos Sprague-Dawley , Transplante Heterotópico/métodos , Veia Cava Inferior/cirurgia , Veia Cava Superior/cirurgiaRESUMO
OBJECTIVE: To determine the role of the terminal complement complex (TCC) in the development of experimental antigen-induced arthritis (AIA) and the therapeutic effects of human anti-C5 single-chain Fv (scFv). METHODS: Two different anti-C5 scFv, one that inhibits both release of C5a and assembly of the TCC (TS-A 12/22) and another that selectively blocks formation of the TCC (TS-A 8), were injected at the onset of AIA. The effects of these scFv on disease severity were evaluated for up to 21 days and compared with the effects of injection of an unrelated scFv. AIA was also established in C6-deficient and C6-sufficient PVG rats to obtain further information on the role of the TCC in this model. RESULTS: TS-A 12/22 and TS-A 8 proved to be equally effective in reducing joint swelling, cell counts and tumor necrosis factor alpha levels in synovial lavage fluids, and the degree of histomorphologic changes compared with the effects of the unrelated scFv. TS-A 12/22 and TS-A 8 prevented the deposition of C9 but not that of C3, confirming the ability of the 2 scFv to neutralize C5. Administration of the 2 anti-C5 scFv after AIA onset also reduced disease severity. In C6-deficient rats with AIA, disease activity was reduced markedly compared with that in C6-sufficient rats. CONCLUSION: These 2 human anti-C5 scFv could represent potential therapeutic reagents to be used in patients with rheumatoid arthritis. In addition, the finding that TS-A 8 was as effective as TS-A 12/22 in reducing disease severity suggests that the TCC is mainly responsible for the joint inflammation and damage observed in AIA.