Phosphorylation of complement factor C3 in vivo

Complement factor C3, the central protein of the complement system, was found to be phosphorylated both in EDTA- and heparin-anticoagulated whole blood and in coagulating blood. Complement S protein (vitronectin) was also found to be phosphorylated under these conditions. Further, purified C3 was found to be a phosphoprotein in vivo, containing 0.15 mol of alkali-labile phosphate/mol of protein. The ATP concentration in plasma was measured and found to be about 2 microM.

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Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells

Gene Therapy ◽

10.1038/s41434-021-00239-9 ◽

2021 ◽

Author(s):

Anna K. Dreismann ◽

Michelle E. McClements ◽

Alun R. Barnard ◽

Elise Orhan ◽

Jane P. Hughes ◽

...

Keyword(s):

Complement System ◽

Functional Expression ◽

Geographic Atrophy ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Factor I ◽

Complement Factor I ◽

Age Related ◽

The Complement System

AbstractDry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

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A novel approach to immunoapheresis of C3a/C3 and proteomic identification of associates

PeerJ ◽

10.7717/peerj.8218 ◽

2019 ◽

Vol 7 ◽

pp. e8218

Author(s):

Wolfgang Winnicki ◽

Peter Pichler ◽

Karl Mechtler ◽

Richard Imre ◽

Ines Steinmacher ◽

...

Keyword(s):

Complement System ◽

Proteomic Analysis ◽

Methodological Approach ◽

Central Component ◽

Complement Factor ◽

Immunoaffinity Column ◽

Novel Approach ◽

Human Disorders ◽

Complement Factor C3 ◽

The Complement System

Background Complement factor C3 represents the central component of the complement cascade and its activation split product C3a plays an important role in inflammation and disease. Many human disorders are linked to dysregulation of the complement system and alteration in interaction molecules. Therefore, various therapeutic approaches to act on the complement system have been initiated. Methods and Results Aiming to develop a tool to eliminate C3a/C3 from the circulation, in a first step a high affine murine monoclonal antibody (mAb) (3F7E2-mAb) was generated against complement factor C3 and selected for binding to the C3a region to serve as immunoaffinity reagent. Functional testing of the 3F7E2-mAb revealed an inhibition of Zymosan-induced cleavage of C3a from C3. Subsequently, a C3a/C3 specific 3F7E2-immunoaffinity column was developed and apheresis of C3a/C3 and associates was performed. Finally, a proteomic analysis was carried out for identification of apheresis products. C3a/C3 was liberated from the 3F7E2-column together with 278 proteins. C3a/C3 interaction specificity was validated by using a haptoglobin immunoaffinity column as control and biostatistic analysis revealed 39 true C3a/C3 interactants. Conclusion A novel and functionally active mAb was developed against complement factor C3a/C3 and used in a specific immunoaffinity column that allows apheresis of C3a/C3 and associates and their identification by proteomic analysis. This methodological approach of developing specific antibodies that can be used as immunoaffinity reagents to design immunoaffinity columns for elimination and further identification of associated proteins could open new avenues for the development of tailored immunotherapy in various complement-mediated or autoimmune diseases.

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POTENSI 6-AZA-INDAZOLE SEBAGAI TERAPI YANG DAPAT DIPERBAIKI UNTUK PERAWATAN RETINOPATI DIABETIK MELALUI INHIBISI FAKTOR D PENYELESAIAN

Al-Iqra Medical Journal : Jurnal Berkala Ilmiah Kedokteran ◽

10.26618/aimj.v3i1.2747 ◽

2019 ◽

Vol 2 (1) ◽

pp. 30-37

Author(s):

Dhitta Shabrina ◽

Ihya Fakrurizal ◽

Afid Brilliana P

Keyword(s):

Diabetic Retinopathy ◽

Complement System ◽

Alternative Pathway ◽

Medical Personnel ◽

Significant Inhibition ◽

Complement Factor ◽

In Vivo Test ◽

Clinical Phase ◽

The Complement System

Diabetes mellitus is a chronic disease that has a high prevalence in Indonesia. One of diabetes’ complications is Diabetic Retinopathy (DR). DR is one of the highest cause of preventable blindness in the world. The treatment for DR up till then uses intravitreal anti-VEGF injection, intravitreal steroid antiinflammation injection. Intravitreal administration can cause some side effects such as increased intraocular pressure and endophthalmitis. Furthermore, patient’s convenience is also disturbed, for always having to see a medical personnel frequently. One pathogenesis of DR is the activation of the complement system which causes lysis of endothelial cells and results in ischemic damage to the retina. This ischemic effect will stimulate VEGF secretion that manifests in the phase of Proliferative Diabetic Retinopathy (PDR). Inhibitor of D factor, 6-aza indazole is a small size protein, less than 300 Da which has the potential to inhibit progress of DR by interfering with the activation of alternative pathway (AP) of the complement system. This is supported by the in-vivo test result, where there is a significant inhibition of intraocular AP activation continuously for 8 hours after an oral administration of this agent at a dose of 30 mg/kg. But this study is still in a pre-clinical phase that uses mice as the subject. Therefore, there must be a further study and clinical trial to find out the dosage and safety of this agent to be applied to humans. Keywords: 6-aza indazole, alternative pathway, complement factor D inhibitor, diabetic retinopathy

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C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue

International Journal of Molecular Sciences ◽

10.3390/ijms22115800 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5800

Author(s):

Philipp Götz ◽

Anna Braumandl ◽

Matthias Kübler ◽

Konda Kumaraswami ◽

Hellen Ishikawa-Ankerhold ◽

...

Keyword(s):

Muscle Tissue ◽

Complement System ◽

Neutrophil Extracellular Trap ◽

Complement Factor ◽

Artery Ligation ◽

Complement Factor C3 ◽

Gastrocnemius Muscles ◽

The Impact ◽

The Complement System ◽

Ischemic Muscle

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31+/BrdU+). Moreover, our results showed that the total number of leukocytes (CD45+) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO+), neutrophil extracellular trap formation (MPO+/CitH3+), and macrophages (CD68+) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68+/MRC1+). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.

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Interaction with complement proteins and dendritic cells implicates LCCL domain-containing proteins (CCps) of malaria parasites in immunomodulation

Biochemical Journal ◽

10.1042/bcj20180494 ◽

2018 ◽

Vol 475 (21) ◽

pp. 3311-3314 ◽

Cited By ~ 1

Author(s):

Puran Singh Sijwali

Keyword(s):

Dendritic Cells ◽

Complement System ◽

Protein Complexes ◽

Immune Defense ◽

Malaria Parasites ◽

Classical Complement Pathway ◽

Soluble Immune Complexes ◽

Multimeric Protein ◽

The Complement System

The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal, Sharma et al. reported that Plasmodiumfalciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system

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Anti-GD2 IgA kills tumors by neutrophils without antibody-associated pain in the preclinical treatment of high-risk neuroblastoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003163 ◽

2021 ◽

Vol 9 (10) ◽

pp. e003163

Author(s):

Mitchell Evers ◽

Marjolein Stip ◽

Kaylee Keller ◽

Hanneke Willemen ◽

Maaike Nederend ◽

...

Keyword(s):

High Risk ◽

Sensory Neurons ◽

Complement System ◽

Severe Pain ◽

Neuroblastoma Cells ◽

Antibody Therapy ◽

Preclinical Treatment ◽

The Complement System

BackgroundThe addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons.MethodsTo reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89).ResultsIgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo.ConclusionsOur results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy.

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