10 research outputs found

    Complement C7 and clusterin form a complex in circulation

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    IntroductionThe complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated.MethodsTo shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum‑purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size‑exclusion chromatography. ResultsProtein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. DiscussionClusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade

    Antioxidants N-Acetylcysteine and Vitamin C Improve T Cell Commitment to Memory and Long-Term Maintenance of Immunological Memory in Old Mice

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    Aging is characterized by reduced immune responses, a process known as immunosenescence. Shortly after their generation, antigen-experienced adaptive immune cells, such as CD8+ and CD4+ T cells, migrate into the bone marrow (BM), in which they can be maintained for long periods of time within survival niches. Interestingly, we recently observed how oxidative stress may negatively support the maintenance of immunological memory in the BM in old age. To assess whether the generation and maintenance of immunological memory could be improved by scavenging oxygen radicals, we vaccinated 18-months (old) and 3-weeks (young) mice with alum-OVA, in the presence/absence of antioxidants vitamin C (Vc) and/or N-acetylcysteine (NAC). To monitor the phenotype of the immune cell population, blood was withdrawn at several time-points, and BM and spleen were harvested 91 days after the first alum-OVA dose. Only in old mice, memory T cell commitment was boosted with some antioxidant treatments. In addition, oxidative stress and the expression of pro-inflammatory molecules decreased in old mice. Finally, changes in the phenotype of dendritic cells, important regulators of T cell activation, were additionally observed. Taken together, our data show that the generation and maintenance of memory T cells in old age may be improved by targeting oxidative stress

    Additional file 2: Figure S1. of Booster vaccination against tetanus and diphtheria: insufficient protection against diphtheria in young and elderly adults

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    Gating strategy for the analysis of the intracellular stainings. A representative example of CD4+ memory T cells producing IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-17, IL-21, TGF-β and GM-CSF after 6 h of tetanus toxoid (10 μg/ml) stimulation is shown (TIF 865 kb

    Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

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    Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis

    Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections

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    Overactivation of the complement system has been characterized in severe COVID-19 cases. Complement components are known to trigger NETosis via the coagulation cascade and have also been reported in human tracheobronchial epithelial cells. In this longitudinal study, we investigated systemic and local complement activation and NETosis in COVID-19 patients that underwent mechanical ventilation. Results confirmed significantly higher baseline levels of serum C5a (24.5 ± 39.0 ng/mL) and TCC (11.03 ± 8.52 µg/mL) in patients compared to healthy controls (p p 6 neutrophils/mL) compared to healthy controls (0.82 (±0.74) × 106 neutrophils/mL) (p < 0.0001). In tracheal fluid, baseline TCC levels but not C5a and NETosis, were significantly higher in patients. Kinetic studies of systemic complement activation revealed markedly higher levels of TCC and CRP in nonsurvivors compared to survivors. In contrast, kinetic studies showed decreased local NETosis in tracheal fluid but comparable local complement activation in nonsurvivors compared to survivors. Systemic TCC and NETosis were significantly correlated with inflammation and coagulation markers. We propose that a ratio comprising systemic inflammation, complement activation, and chest X-ray score could be rendered as a predictive parameter of patient outcome in severe SARS-CoV-2 infections

    DataSheet_1_Complement C7 and clusterin form a complex in circulation.docx

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    IntroductionThe complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated.MethodsTo shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum‑purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size‑exclusion chromatography. ResultsProtein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. DiscussionClusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade. </p
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