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1.
Med Microbiol Immunol ; 213(1): 8, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38767707

RESUMO

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.


Assuntos
Proteínas do Sistema Complemento , Modelos Animais de Doenças , Lipopolissacarídeos , Antígenos O , Salmonella enteritidis , Salmonella enteritidis/imunologia , Salmonella enteritidis/patogenicidade , Animais , Antígenos O/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Lipopolissacarídeos/imunologia , Evasão da Resposta Imune , Viabilidade Microbiana , Mariposas/microbiologia , Mariposas/imunologia , Virulência , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Ativação do Complemento , Lepidópteros/imunologia , Lepidópteros/microbiologia
2.
Biotechnol J ; 17(6): e2100213, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35174641

RESUMO

BACKGROUND AND AIM: Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. MAIN METHODS AND RESULTS: We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. CONCLUSIONS AND IMPLICATIONS: Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.


Assuntos
Análise de Célula Única , Transcriptoma , Anticorpos/genética , Perfilação da Expressão Gênica , Oligonucleotídeos/genética , Proteômica , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
3.
Thromb Res ; 208: 129-137, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34768097

RESUMO

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. OBJECTIVE: To assess antibodies in interaction with the activation of platelets and complement triggered by VITT. METHODS: Antibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. RESULTS: None of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. CONCLUSIONS: Our findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.


Assuntos
Vacinas contra Adenovirus , COVID-19 , Adenoviridae , Plaquetas , Vacinas contra COVID-19 , Humanos , Imunoglobulina G , Fator Plaquetário 4 , SARS-CoV-2
4.
Semin Immunol ; 45: 101337, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31757607

RESUMO

Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.


Assuntos
Proteínas do Sistema Complemento/imunologia , Tolerância Imunológica , Imunomodulação , Animais , Apoptose , Autoimunidade , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , Gravidez , Receptores de Complemento/metabolismo
5.
Kidney Int ; 94(6): 1141-1150, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30322716

RESUMO

Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3.


Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Ativação do Complemento/imunologia , Complemento C3/imunologia , Properdina/imunologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Complemento C3/genética , Complemento C3/metabolismo , Modelos Animais de Doenças , Feminino , Membrana Basal Glomerular/citologia , Membrana Basal Glomerular/imunologia , Membrana Basal Glomerular/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Properdina/genética , Properdina/metabolismo , Ligação Proteica/imunologia
6.
Transplantation ; 102(1): 79-87, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28731906

RESUMO

BACKGROUND: Kidneys derived from brain-dead (BD) donors have lower graft survival rates compared with kidneys from living donors. Complement activation plays an important role in brain death. The aim of our study was therefore to investigate the effect of C1-inhibitor (C1-INH) on BD-induced renal injury. METHODS: Brain death was induced in rats by inflating a subdurally placed balloon catheter. Thirty minutes after BD, rats were treated with saline, low-dose or high-dose C1-INH. Sham-operated rats served as controls. After 4 hours of brain death, renal function, injury, inflammation, and complement activation were assessed. RESULTS: High-dose C1-INH treatment of BD donors resulted in significantly lower renal gene expression and serum levels of IL-6. Treatment with C1-INH also improved renal function and reduced renal injury, reflected by the significantly lower kidney injury marker 1 gene expression and lower serum levels of lactate dehydrogenase and creatinine. Furthermore, C1-INH effectively reduced complement activation by brain death and significantly increased functional levels. However, C1-INH treatment did not prevent renal cellular influx. CONCLUSIONS: Targeting complement activation after the induction of brain death reduced renal inflammation and improved renal function before transplantation. Therefore, strategies targeting complement activation in human BD donors might clinically improve donor organ viability and renal allograft survival.


Assuntos
Morte Encefálica , Proteína Inibidora do Complemento C1/uso terapêutico , Nefropatias/prevenção & controle , Transplante de Rim , Animais , Ativação do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Interleucina-6/análise , Masculino , Ratos , Ratos Endogâmicos F344
7.
FASEB J ; 31(7): 3193-3204, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28396344

RESUMO

The complement system, and specifically C5a, is involved in renal ischemia-reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild-type, C5aR1-/-, and C5aR2-/- mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2-/- mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an in vivo migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2-/- mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell-expressed C5aR2 vs leukocyte-expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell-expressed C5aR2 and leukocyte-expressed C5aR2 mediate IR-induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.-Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.-L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia-reperfusion injury.


Assuntos
Nefropatias/etiologia , Receptor da Anafilatoxina C5a/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Movimento Celular/fisiologia , Regulação da Expressão Gênica , Leucócitos/fisiologia , Camundongos , Camundongos Knockout , Ativação de Neutrófilo , Neutrófilos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor da Anafilatoxina C5a/genética
8.
Mol Immunol ; 76: 13-21, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27337595

RESUMO

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9.


Assuntos
Ativação do Complemento/imunologia , Complemento C3/imunologia , Complemento C6/imunologia , Complemento C9/imunologia , Caracteres Sexuais , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
9.
J Immunol Methods ; 419: 25-34, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25733354

RESUMO

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.


Assuntos
Ativação do Complemento/imunologia , Complemento C3b/imunologia , Complemento C3c/imunologia , Rim/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Ativação do Complemento/genética , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C3c/genética , Complemento C3c/metabolismo , Complemento C9/imunologia , Complemento C9/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Estudos de Viabilidade , Rim/irrigação sanguínea , Rim/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reprodutibilidade dos Testes , Fatores de Tempo
10.
Chemistry ; 16(23): 6912-8, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20437425

RESUMO

Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.


Assuntos
Moléculas de Adesão Celular/química , DNA/química , Dendrímeros/química , Proteínas/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Ligação Proteica , Raios Ultravioleta
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