Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice.

ABSTRACT: The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a "TA" nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.

SUBMITTER: Wang Q

PROVIDER: S-EPMC4547999 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

A novel role for the fifth component of complement (C5) in cardiac physiology.

Project description:We have previously demonstrated that C5-deficient A/J and recombinant congenic BcA17 mice suffer from cardiac dysfunction when infected with C. albicans blastospores intravenously. During these studies we had observed that, even in the control un-infected state, BcA17 hearts displayed alterations in gene expression that have been associated with pathological cardiac hypertrophy in comparison to parental C5-sufficient C57Bl/6J (B6) mice. Of note was an increase in the expression of Nppb, a member of the fetal gene program and a decrease in the expression of Rgs2, an inhibitor of the hypertrophic response. We now report that C5-deletion has also affected the expression of other elements of the fetal gene program. Moreover deleting the C5a receptor, C5aR, has essentially the same effect as deleting C5, indicating a key role for C5a-C5aR signaling in the phenotype. Having noted a pathological phenotype in the un-infected state, we investigated the role of C5 in the response to cardiac stress. In previous studies, comparison of the expression profiles of C. albicans-infected BcA17 and similarly infected B6 hearts had revealed a paucity of cardioprotective genes in the C5-deficient heart. To determine whether this was also directly linked to C5-deficiency, we tested the expression of 5 such genes in the C. albicans-infected C5aR(-/-) mice. We found again that deletion of C5aR recapitulated the alterations in stress response of BcA17. To determine whether our observations were relevant to other forms of cardiac injury, we tested the effect of C5-deficiency on the response to isoproterenol-induced hypertrophic stimulation. Consistent with our hypothesis, A/J, BcA17 and C5aR(-/-) mice responded with higher levels of Nppa expression than B6 and BALB/c mice. In conclusion, our results suggest that an absence of functional C5a renders the heart in a state of distress, conferring a predisposition to cardiac dysfunction in the face of additional injury.

| S-EPMC3148243 | biostudies-literature

Inhibition of complement pathway activation with Pozelimab, a fully human antibody to complement component C5.

Project description:Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.

| S-EPMC7209288 | biostudies-literature

Complement C5-deficient mice are protected from seizures in experimental cerebral malaria.

Project description:Studies have demonstrated that the membrane attack complex (MAC) of complement can evoke seizures when injected directly into rodent brain. In the course of studies that examine the role of complement in the development of experimental cerebral malaria (ECM), we observed fewer seizures in mice deficient in C5, a component required for MAC formation. To determine if the MAC contributed to the tonic-clonic seizures characteristic of ECM, we performed long-term video-electroencephalography (EEG) on C5(-/-) mice with Plasmodium berghei ANKA-induced cerebral malaria and observed significantly reduced spike and seizure frequency compared to wild-type mice. Our data suggest a role for the MAC in malaria-induced seizures and that inhibition of the terminal complement pathway may reduce seizures and seizure-related neurocognitive deficits.

| S-EPMC4285666 | biostudies-literature

Complement Component C5 and TLR Molecule CD14 Mediate Heme-Induced Thromboinflammation in Human Blood.

Project description:Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1α, IFN-γ, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (p < 0.05 for all) reduced the upregulation of CD11b on granulocytes and monocytes by 59 and 40%, respectively (p < 0.05), and attenuated monocytic tissue factor expression by 33% (p < 0.001). Blocking CD14 attenuated IL-6 and TNF by more than 50% (p < 0.05). In contrast to single inhibition, combined C5 and CD14 was required for a significantly attenuated prothrombin cleavage (72%, p < 0.05). Markers of thromboinflammation were also quantified in two patients admitted to the hospital with sickle cell disease (SCD) crisis. Both SCD patients had pronounced hemolysis and depleted plasma hemopexin and haptoglobin. Plasma heme and complement activation was markedly increased in one patient, a coinciding observation as demonstrated ex vivo. In conclusion, heme-induced thromboinflammation was largely attenuated by C5 inhibition alone, with a beneficial effect of adding a CD14 inhibitor to attenuate prothrombin activation. Targeting C5 has the potential to reduce thromboinflammation in SCD crisis patients.

| S-EPMC6731449 | biostudies-literature

The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.

Project description:The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.

| S-EPMC1163403 | biostudies-other

Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans.

Project description:Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5+/+) and congenic C57BL/6 (B6 C5-/-, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5-/- mice when compared to B6 C5+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5+/+ mice than in B6 C5-/- mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5+/+ than B6 C5-/- mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5+/+ and B6 C5-/- were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.

| S-EPMC5852101 | biostudies-literature

Soluble MAC is primarily released from MAC-resistant bacteria that potently convert complement component C5.

Project description:The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

| S-EPMC9402229 | biostudies-literature

Neutralization of complement component C5 ameliorates the development of dextran sulfate sodium (DSS)-colitis in mice.

Project description:The complement system is a potent effector of innate immunity. To elucidate the pathophysiological role of the complement system in inflammatory bowel disease, we evaluated the effects of anti-C5 antibodies on the development of dextran sulfate sodium-induced colitis in mice. Dextran sulfate sodium-colitis was induced in BALB/c mice with intraperitoneal administrations of anti-C5 antibodies (1 mg/body [DOSAGE ERROR CORRECTED]) every 48 h. Tissue samples were evaluated by standard histological procedures. The mucosal mRNA expression of the inflammatory cytokines was analyzed by real-time PCR. Body weight loss in the mice was completely blocked by the administration of anti-C5 antibody. The disease activity index was significantly lower in the anti-C5 antibody-treated mice than the dextran sulfate sodium mice. The colonic weight/length ratio, histological colitis score and mucosal myeloperoxidase activity were significantly lower in the anti-C5 antibody-treated mice than the dextran sodium sulfate mice. The administration of the anti-C5 antibody significantly reduced the mucosal expression of mRNAs for tumor necrosis factor-?, interleukin-1? and interleukin-6. In conclusion, the complement system plays a role in the development of dextran sodium sulfate-induced experimental colitis.

| S-EPMC3541422 | biostudies-literature

Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants.

Project description:BackgroundBabies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4α (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.MethodsDroplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies' genotypes were ascertained postnatally by Sanger sequencing.ResultsDroplet digital PCR assays for GCK or HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no false-positive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.ConclusionsThis assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.

| S-EPMC7611030 | biostudies-literature

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

Project description:We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

| S-EPMC1138894 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data