C5a Receptor

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Expression of the C5a receptor (CD88) on granulocytes and monocytes in patients with severe sepsis

Expression of the C5a receptor (CD88) on granulocytes and monocytes in patients with severe sepsis

The samples were analysed on an EPICS-PROFILE II flow cytometer (Coulter Company Inc, Hialeah, FL, USA). The analysis used a fixed protocol, with the same settings main- tained for forward scatter and side scatter throughout the study. The fluorescence was calibrated daily, to compensate for the variation of the signals from the flow cytometer, using standardised beads (Flow Set; Coulter Company Inc). The granulocytes and monocytes were separated on the basis of their forward scatter and side scatter patterns, and the staining with anti-CD14 was used to check the identification of the monocytes. Gates were set around the granulocyte and monocyte populations, and the FITC fluorescence within the gates was measured. A minimum of 10,000 events in the granulocyte gate was counted. The granulocyte and monocyte expression of the C5a receptor (CD88) was measured as specific mean fluorescence intensity of the whole population of granulocytes and monocytes, and as the relative amount of CD88-positive granulocytes and monocytes. The specific mean fluorescence intensity of the granulocyte and monocyte C5a receptor expression was calculated by subtracting the background mean fluorescence intensity obtained with the negative isotype control mAb from the value obtained with the anti-CD88 mAb. The relative amounts of C5a receptor-posi- tive granulocytes or monocytes were calculated as the relative numbers of granulocytes or monocytes, respectively, showing a higher fluorescence intensity when stained with the anti- CD88 mAb than with the negative control mAb.
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C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA

C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA

Anaphylatoxin C5a is a strong chemoattractant and an important factor of the complement system [11, 12]. The C5a receptor (C5aR) is a G-protein coupled receptor expressed by leukocytes [13]. C5a promotes leukocyte migration and their production of radical oxygen species by binding to the C5aR expressed on their cellular membranes, resulting in the initiation of inflammation [13, 14]. We previously reported that several solid cancer cells also express C5aR on their cellular membrane and that the C5a-C5aR axis promotes the invasiveness of cholangiocarcinoma by inducing actin reorganization and production of matrix metalloproteases [15]. It was also reported that C5aR expression in non-small lung cancer, breast cancer and ovarian cancer was associated with a poor prognosis [16–18]. However, the role of C5aR in GC is still mostly unknown. Here, we investigated the role of C5aR on the motility and invasive ability of GC cells in vitro. Moreover, we analyzed the relationship between C5aR-expression and the prognosis of GC patients.
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Increased C5a receptor expression in sepsis

Increased C5a receptor expression in sepsis

Excessive production of the complement activation product C5a appears to be harmful during the development of sepsis in rodents. Little is known about the role of the C5a receptor (C5aR) and its presence in different organs during sepsis. Using the cecal ligation/puncture (CLP) model in mice, we show here that C5aR immunoreactivity was strikingly increased in lung, liver, kidney, and heart early in sepsis in both control and neutrophil-depleted mice. C5aR mRNA expression in these organs was also significantly increased during sepsis. Immunohistochemical analysis revealed patterns of increased C5aR expression in parenchymal cells in all four organs following CLP. Mice injected at the start of CLP with a blocking IgG to C5aR ( α C5aR) showed dramatically improved survival when com- pared with animals receiving nonspecific IgG, as did mice injected with α C5a. In α C5aR-treated mice, serum levels of IL-6 and TNF-α and bacterial counts in various organs were significantly reduced dur- ing CLP when compared with control CLP animals. These studies demonstrate for the first time that C5aR is upregulated in lung, liver, kidney, and heart during the early phases of sepsis and that block- ade of C5aR is highly protective from the lethal outcome of sepsis.
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Complement C5a Receptor 1 Exacerbates the Pathophysiology of N  meningitidis Sepsis and Is a Potential Target for Disease Treatment

Complement C5a Receptor 1 Exacerbates the Pathophysiology of N  meningitidis Sepsis and Is a Potential Target for Disease Treatment

ABSTRACT Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly pro- gressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory re- sponse is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate im- mune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activa- tion concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed suscepti- bility that was enhanced by ⬎ 1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1 ⫺ / ⫺ mice
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An unexpected role for the anaphylatoxin C5a receptor in allergic sensitization

An unexpected role for the anaphylatoxin C5a receptor in allergic sensitization

The anaphylatoxins complement component 3a and 5a (C3a and C5a, respec- tively) are classically seen as proinflammatory mediators of allergic asthma that recruit inflammatory cells, induce edema, and cause bronchoconstric- tion. A few years ago, controversy arose when it was shown that C5-deficient mice were more susceptible to experimental asthma compared with C5-suf- ficient mice. In a study by Köhl et al. in this issue of the JCI, it is shown in a series of truly “complementary” experiments that C5a receptor (C5aR) blockade promotes Th2 sensitization upon first exposure to inhaled aller- gen, whereas C5aR blockade during established inflammation suppresses the cardinal features of asthma (see the related article beginning on page 783). Blockade of C5aR alters the function of airway DCs, crucial for induc- ing and maintaining Th2 responses in the lung. Targeting C5aR as a treat- ment for established asthma could be beneficial, but might be accompanied by sensitization to novel antigens.
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Inhibition of complement C5a prevents breakdown of the blood brain barrier and pituitary dysfunction in experimental sepsis

Inhibition of complement C5a prevents breakdown of the blood brain barrier and pituitary dysfunction in experimental sepsis

Encephalopathy syndrome is a well described complication of sepsis in the ICU. This phenomenon is thought to represent a consequence of inflammation-mediated dysfunction of the blood-brain barrier (BBB), thus allowing neurotoxic mediators to extravasate from the peripheral circulation into the sub- arachnoid space or into the brain parenchyma. Noteworthy, the focus of research studies have only addressed in more depth the neuro-inflammatory and metabolic intracerebral changes in sepsis [23-29]. The complement anaphylatoxin C5a has been characterised as a mediator of BBB dysfunction in a variety of central nervous system (CNS) disorders, includ- ing traumatic brain injury and bacterial meningitis [30-32]. In addition, the detection of the C5a receptor (C5aR) on neurons and the observed upregulation of neuronal C5aR expression under inflammatory conditions [31,33-35] renders the brain more vulnerable to C5a-mediated neuropathophysiological sequelae secondary to a disruption of the BBB [30,31,36,37]. The complement cascade has only recently been implicated in the pathophysiology of septic encephalopathy [38].
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Complement-targeted therapy: development of C5- and C5a-targeted inhibition

Complement-targeted therapy: development of C5- and C5a-targeted inhibition

and C5a receptor antagonists may be efficacious at treat- ing various inflammatory diseases involving complements. There is interest in the efficacy of these anti-complement agents on RA. In fact, numerous findings have suggested a relationship between RA and C5 and C5a receptors. C5a levels in synovial fluid are elevated in patients with RA [16], a genome-wide association study (GWAS) indicated that the TRAF1-C5 region is related to RA in humans [17, 18], numerous animal models have indicated that C5 is a gene responsible for causing arthritis [19, 20], and C5 and C5a receptor knockout mice are resistant to arthritis [21, 22]. However, clinical trials indicated that eculizumab (a hu- manized anti-C5 monoclonal antibody) and PMX53 and MP-435 (C5a receptor antagonists) had little efficacy in treating RA [23, 24].
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The complement system in glioblastoma multiforme

The complement system in glioblastoma multiforme

AP: Alternative Pathway; BBB: Blood Brain Barrier; bFGF: basic Fibroblast Growth Factor; C1-INH: C1-inhibitor; C3aR: C3a Receptor; C5aR1: C5a Receptor-1; C5b-9: Membrane Attack Complex; cC1qR: collagen C1q Receptor (Calreticulin); CNS: Central Nervous System; CREB: Cellular Transcription Factor; CSF 1: Colony Stimulating Factor 1; CXCR1: C-X-C Motif Chemokine Receptor 1; CXCR2: C-X-C Motif Chemokine Receptor 2; CXCR4: C-X-C Motif Chemokine Receptor 4; DAMP: Damage-Associated Molecular Pattern; EC: Endothelial Cells; eNOS: endothelial Nitric Oxide synthase; ERK1/2: Extracellular-signal regulated kinases; Fz: Frizzled; GBM: Glioblastoma Multiforme; gC1qR: Globular C1q Receptor; GSC: Glioma Stem-like Cell; HIF: Hypoxia Inducible Factor; HMEC: Human Mammary Epithelial Cells; hPSC: human induced Pluripotent Stem Cells; HVEC: Human Umbilical Vein Endothelial Cells; HVEC-d: Human Dermal Microvascular Endothelial Cells; IFN γ : Interferon- γ ; LPS: lipopolysaccharide; LRP6: Low-
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The C5a anaphylatoxin receptor CD88 is expressed in presynaptic terminals of hippocampal mossy fibres

The C5a anaphylatoxin receptor CD88 is expressed in presynaptic terminals of hippocampal mossy fibres

The anaphylatoxin C5a, a 74 amino acid glycoprotein, functions primarily as a pro-inflammatory mediator [3,4]. In the periphery, the release of C5a results in a host of inflammatory responses, including increased vascular per- meability, chemotaxis of inflammatory cells, and the release of cytokines and chemokines [11]. These responses are primarily mediated via a C5a-selective seven-transmembrane G-protein coupled receptor (termed CD88) [12]. This C5a-receptor is expressed on a wide range of peripheral cells, including neutrophils, monocytes, activated mast cells, endothelial cells, and vas- cular smooth muscle cells [12]. In the CNS, CD88 can be found on astrocyctes, microglia and neurons in normal human and mouse brains [10,13-19]. Neuronal expres- sion of CD88 has been reported within the cornus ammonis sub-fields (CA1 - 3) of the hippocampus, the dentate gyrus, the neocortex, and the cerebellum [18]. Similarly, in situ hybridization has demonstrated CD88 mRNA within neurons of the neocortex, cerebellum and dentate gyrus [18].
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The interaction between C5a and sphingosine 1 phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation

The interaction between C5a and sphingosine 1 phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation

AAV: antineutrophil cytoplasmic antibody-associated vasculitis; ANCA: antineutrophil cytoplasmic antibody; BSA: bovine serum albumin; BVAS: Birmingham vasculitis activity score; C5aR: C5a receptor; C5L2: C5a receptor-like 2; DHR: dihydrorhodamine; EDTA: ethylene diamine tetraacetic acid; EGPA: eosinophilic granulomatosis with polyangiitis; ELISA: enzyme- linked immunosorbent assay; FACS: fluorescence activated cell sorting; FMLP: N-formyl-methionyl-leucyl-phenylalanine; GPA: granulomatosis with polyangiitis; HBSS: Hank ’ s balanced salt solution; HRP: horseradish peroxidase; Ig: immunoglobulin; LPS: lipopolysaccharide; MFI: mean fluorescence intensity; MPA: microscopic polyangiitis; MPO: myeloperoxidase; OD: optical density; PDGF: platelet-derived growth factor; PE: phycoerythrin; PR3: proteinase-3; S1P: sphingosine-1-phosphate; Sphk: sphingosine kinase; TMB: 3,3 ′ -5,5 ′ tetramethylbenzidin; TNF: tumor necrosis factor; TNFR1: tumor necrosis factor receptor 1.
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Neuroprotective effects of intravenous immunoglobulin are mediated through inhibition of complement activation and apoptosis in a rat model of sepsis

Neuroprotective effects of intravenous immunoglobulin are mediated through inhibition of complement activation and apoptosis in a rat model of sepsis

There is increasing evidence that innate immunity disturbance in sepsis-induced organ dysfunction might be linked to the uncontrolled activation of the complement system [13, 14]. Also, C5a and C5a receptor expression levels were reported to be ele- vated in the lung, liver, kidney, pituitary gland, and heart during sepsis [5, 15]. In our study, significantly elevated levels of C5a were also found in the brain samples of the rats with sepsis. Likewise, C5a receptor expression levels showed a marked increase in all CLP groups on day 1 and remained high in the CLP group with no treatment. Al- though systemic complement activity was reduced in the IVIg-treated rats, cerebral complement and complement inhibitor factor levels were mostly comparable among treated and non-treated groups. As an exception, the IVIg-treated rats showed a strik- ing downregulation of C5a and C5a receptor on day 10. Our results suggest that similar to other sepsis-afflicted tissues, C5a plays a major role in sepsis-induced destruction of the nervous system and apparently IVIg treatment reduces complement-based ana- phylatoxin activity and ameliorates septic encephalopathy presumably through this mechanism of action.
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C5a2 can modulate ERK1/2 signaling in macrophages via heteromer formation with C5a1 and beta-arrestin recruitment

C5a2 can modulate ERK1/2 signaling in macrophages via heteromer formation with C5a1 and beta-arrestin recruitment

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein coupled receptors (GPCR); namely the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, up-regulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte derived macrophages (HMDM). A bioluminescence resonance energy transfer (BRET) assay was used to assess recruitment of -arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated -arrestin 2 recruitment levels in comparison to C5a-des Arg, while no significant difference was observed at C5a2. A constitutive complex that formed between -arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for -arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was
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Prevention of C5aR1 signaling delays microglial inflammatory polarization, favors clearance pathways and suppresses cognitive loss

Prevention of C5aR1 signaling delays microglial inflammatory polarization, favors clearance pathways and suppresses cognitive loss

All animal experimental procedures were reviewed and ap- proved by the Institutional Animal Care and Use Commit- tee of University of California at Irvine, and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The AD mouse model used was the Arctic48, which carry the human APP transgene with the Indiana (V717F), Swedish (K670 N + M671 L), and Arctic (E22G) mutations (under the control of the platelet- derived growth factor-ß promoter), and thus produce Aß protofibrils and fibrils as early as 2–4 months old [30], gra- ciously provided by Dr. Lennart Mucke (Gladstone Insti- tute, San Francisco, CA, USA). C5aR1 knockout mice generated by target deletion of the C5a receptor gene [32], were crossed with Arctic +/ − mice to produce Arctic mice lacking C5aR1 (Arctic/C5aR1KO) and wild type littermate mice lacking the C5a receptor (C5aR1KO) that were assessed for behavioral deficits and pathology in compari- son to the C5aR1 sufficient animals. To enable isolation of microglia distinct from any infiltrating myeloid cells for differential gene expression studies, homozygous breeding pairs of CX3CR1-GFP and CCR2-RFP mice were obtained from Jackson Laboratories and bred to produce double homozygous reporter mice (CX3CR1 GFP/GFP CCR2 RFP/RFP ). Double homozygous reporter mice were bred with Arctic
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Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo

Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo

Neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mech- anism in neurodegenerative disorders. Excitation result- ing from stimulation of the ionotropic glutamate receptors is known to cause neuronal apoptosis. Kainic acid (KA) is an agonist for a subtype of ionotropic gluta- mate receptor, and administration of KA has been shown to increase production of reactive oxygen species, mito- chondrial dysfunction, and apoptosis in neurons in brain [10]. We had earlier reported that the complement com- ponent C5 neuroprotects against excitotoxicity; further we showed that mice genetically deficient of complement component C5 revealed a higher susceptibility to KA neu- rodegeneration [11,12] suggesting that in addition to their pro-inflammatory mechanisms, specific comple- ment components may also mediate neuroprotection. This hypothesis was further supported by evidence show- ing that C5a may neuroprotect against glutamate medi- ated apoptosis through the regulation of mitogen activated protein kinase (MAPK) signal transduction path- ways [13,14] or by inhibition of caspase-3 activity [15]. Based on the evidence that neuronal death in response to excitotoxic insult involves the regulation of GluR2 recep- tor expression [16] and that GluR2 receptor expression is reduced coincidental to increase in expression of apop- totic markers like caspase 3 in Alzheimer's brain [17] we decided to explore the role of GluR2 receptors in C5a mediated protection in vivo and in vitro. In the present study using C5a receptor knockout (C5aRKO) mice we found that neurons were more susceptible to excitotoxic- ity resulting in apoptotic injury in the absence of the C5a receptor. Our study suggests that C5a may protect against neurodegenerative excitotoxicity and apoptosis in neuro- nal cells through the regulation of GluR2 receptor expres- sion in vitro and in vivo.
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Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis

Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis

Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor–deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis.
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C5a induced expression of P selectin in endothelial cells

C5a induced expression of P selectin in endothelial cells

superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and
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Derivation of ligands for the complement C3a receptor from the C-terminus of C5a

Derivation of ligands for the complement C3a receptor from the C-terminus of C5a

Reis, E.S., Chen, H., Sfyroera, G., Monk, P.N., Kohl, J., Ricklin, D., Lambris, J.D., 2012. C5a receptor-dependent cell activation by physiological concentrations of desarginated C5a: insights from a novel label-free cellular assay. Journal of immunology 189, 4797-4805. Schofield, Z.V., Woodruff, T.M., Halai, R., Wu, M.C., Cooper, M.A., 2013. Neutrophils-a key component of ischemia-reperfusion injury. Shock 40, 463-470.

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In vivo clearance and tissue distribution of C5a and C5a des arginine complement fragments in rabbits

In vivo clearance and tissue distribution of C5a and C5a des arginine complement fragments in rabbits

response observed with both mediators. Clearance of the mediators was primarily seen in the highly vascularized organs: the lung, spleen, liver, and kidney. A time-dependent accumulation was seen initially in the lung, followed by the spleen, liver, and kidney. Histologic examination showed a marked increase in the number of neutrophils within the lung and spleen. Depletion of circulating neutrophils by nitrogen mustard pretreatment of rabbits showed no change in the amount of labeled mediator bound in the lung, whereas splenic accumulation was dependent on the presence of neutrophils. These results indicate that C5a […]
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Effects of anti C5a antibodies on the adult respiratory distress syndrome in septic primates

Effects of anti C5a antibodies on the adult respiratory distress syndrome in septic primates

develop decreased oxygenation (P less than 0.05) or increased extravascular lung water (P less than 0.05). They also had a marked recovery in their mean arterial blood pressure (P less than 0.05). This study demonstrates that treatment with rabbit anti-human C5a des arg antibodies attenuates ARDS and some of the systemic manifestations of sepsis in

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Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release

Differential effects of the complement peptides, C5a and C5a des Arg on human basophil and lung mast cell histamine release

induce mast cell leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) release. LTC4 release was also negligible from basophils where C5a was a potent histamine release stimulus. Supernatants from C5a-challenged mast cells remained fully active on basophils, excluding carboxypeptidase inactivation of C5a as an explanation for the lung mast cell results. In contrast to lung, skin mast cells were C5a-responsive (histamine release = 8 +/- 1%, at 55 micrograms/ml, n = 2). We conclude that C5a, though devoid of activity on the human lung mast cell, is a human basophil and skin mast cell secretagogue. These findings demonstrate significant organ-specific heterogeneity in mast cell responsiveness.
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