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Biomedical subjects

Alok Agrawal

Publications and source records attributed to Alok Agrawal.

16 recordsLinked to original sources
PubMed2007

Human C-reactive protein protects mice from Streptococcus pneumoniae infection without binding to pneumococcal C-polysaccharide.

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcgamma receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.

Animals↗
PubMed2006

Interaction of C1q with IgG1, C-reactive protein and pentraxin 3: mutational studies using recombinant globular head modules of human C1q A, B, and C chains.

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.

Amino Acid Sequence↗
PubMed2006

Role of the property of C-reactive protein to activate the classical pathway of complement in protecting mice from pneumococcal infection.

C-reactive protein (CRP) is not an acute-phase protein in mice, and therefore, mice are widely used to investigate the functions of human CRP. It has been shown that CRP protects mice from pneumococcal infection, and an active complement system is required for full protection. In this study, we assessed the contribution of CRP's ability of activating the classical pathway of complement in the protection of mice from lethal infection with virulent Streptococcus pneumoniae type 3. We used two CRP mutants, Y175A and K114A. The Y175A CRP does not bind C1q and does not activate complement in human serum. The K114A CRP binds C1q and activates complement more efficiently than wild-type CRP. Passively administered, both CRP mutants and the wild-type CRP protected mice from infection equally. Infected mice injected with wild-type or mutant CRP had reduced bacteremia, resulting in lower mortality and increased longevity compared with mice that did not receive CRP. Thus, the protection of mice was independent of CRP-mediated activation of the classical pathway of complement. To confirm that human CRP does not differentiate between human and mouse complement, we analyzed the binding of human CRP to mouse C1q. Surprisingly, CRP did not react with mouse C1q, although both mutant and wild-type CRP activated mouse C3, indicating species specificity of CRP-C1q interaction. We conclude that the mouse is an unfit animal for exploring CRP-mediated activation of the classical complement pathway, and that the characteristic of CRP to activate the classical complement pathway has no role in protecting mice from infection.

Animals↗
PubMed2005

Regulation of basal and induced expression of C-reactive protein through an overlapping element for OCT-1 and NF-kappaB on the proximal promoter.

C-reactive protein (CRP) is an acute phase protein produced by hepatocytes. A minor elevation in the baseline levels of serum CRP is considered an indicator of chronic inflammation. In hepatoma Hep3B cells, IL-6 induces CRP expression by activating transcription factors STAT3 and C/EBPbeta. IL-1 synergistically enhances the effects of IL-6. The first 157 bp of the CRP promoter are sufficient for IL-1 synergy. Previously, NF-kappaB, a transcription factor activated by IL-1beta in Hep3B cells, has been shown to increase endogenous CRP expression. The purpose of this study was to investigate the possible action of NF-kappaB on the 157 bp of the proximal promoter. In this study we show that NF-kappaB requires and acts synergistically with C/EBPbeta on the CRP-proximal promoter to regulate CRP expression. We located the regulatory element that consisted of overlapping binding sites for NF-kappaB (p50-p50 and p50-p65) and OCT-1. The kappaB site was responsible for the synergy between NF-kappaB and C/EBPbeta and was also necessary for the CRP transactivation by C/EBPbeta through the C/EBP site. Mutation of the kappaB site decreased the synergistic effect of IL-1beta on IL-6-induced CRP expression. Basal CRP expression increased dramatically when binding of both OCT-1 and NF-kappaB was abolished. Combined data from luciferase transactivation assays and EMSA lead us to conclude that the binding of OCT-1 to the promoter, facilitated by p50-p50 in a novel way, represses, whereas replacement of OCT-1 by p50-p65 induces CRP transcription in cooperation with C/EBPbeta. This model for CRP expression favors the variation seen in baseline serum CRP levels in a normal healthy population.

C-Reactive Protein↗
PubMed2005

Statins and nitric oxide reduce C-reactive protein production while inflammatory conditions persist.

C-reactive protein (CRP) is made in liver and its serum concentration increases in inflammation. Measurement of serum CRP is recommended for use as an indicator of inflammation and predictor of atherosclerosis. Cholesterol-lowering drugs statins also lower CRP. To evaluate statin-mediated CRP reduction and to reassess clinical usefulness of CRP, we investigated regulation of CRP gene expression. Here, we show that pravastatin and simvastatin prevent the induction of CRP expression in human hepatoma Hep3B cells exposed to proinflammatory cytokines IL-6 and IL-1beta The nitric oxide (NO) donor, sodium nitroprusside, also prevented the induction of CRP expression while the CRP inducers IL-6 and IL-1beta were present with the cells. The effect of NO on CRP expression was at the level of transcription. These findings suggest that the decrease in CRP level in vivo after statin-treatment does not necessarily reflect absence of inflammation, and that NO-releasing drugs have the potential to reduce serum CRP levels. Thus, the measurement of serum CRP levels alone in individuals on statin/NO-therapy is not as useful as was imagined.

Anticholesteremic Agents↗
PubMed2004

CRP after 2004.

C-reactive protein (CRP) that has been conserved throughout evolution is a host-defense molecule. Its attraction towards phosphocholine-ligands, such as modified low-density lipoprotein, and apoptotic cells leads to the "masking" of these substances that have the capabilities to otherwise engage in deleterious activities. Complement activation by CRP complexes and the modulation by CRP of complement activation by its ligands add up to its beneficial effects. In the presence of CRP, production of membrane-damaging last product of the complement pathway is arrested. CRP is currently serving as an indicator of cardiovascular diseases, but to pinpoint the role of CRP in atherosclerosis, a drug that can lower cholesterol levels, but not the CRP levels, is needed for experimentation.

Animals↗
PubMed2004

Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications.

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+ -bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+ -binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+ -bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+ -bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+ -bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.

Base Sequence↗
PubMed2004

Structural and functional anatomy of the globular domain of complement protein C1q.

C1q is the first subcomponent of the classical pathway of the complement system and a major connecting link between innate and acquired immunity. As a versatile charge pattern recognition molecule, C1q is capable of engaging a broad range of ligands via its heterotrimeric globular domain (gC1q) which is composed of the C-terminal regions of its A (ghA), B (ghB) and C (ghC) chains. Recent studies using recombinant forms of ghA, ghB and ghC have suggested that the gC1q domain has a modular organization and each chain can have differential ligand specificity. The crystal structure of the gC1q, molecular modeling and protein engineering studies have combined to illustrate how modular organization, charge distribution and the spatial orientation of the heterotrimeric assembly offer versatility of ligand recognition to C1q. Although the biochemical and structural studies have provided novel insights into the structure-function relationships within the gC1q domain, they have also raised many unexpected issues for debate.

Animals↗
PubMed2003

The phosphocholine and the polycation-binding sites on rabbit C-reactive protein are structurally and functionally distinct.

C-reactive protein (CRP) is an acute phase protein in humans and rabbits that has the ability to bind a number of biologically important ligands including phosphocholine (PCh), histones, and polycations. In addition to this recognition function, ligand-complexed or aggregated CRP is capable of activating the classical complement pathway. We have generated two strains of transgenic mice in order to study CRP-binding to PCh and consequent complement activation. Based on crystallographic and mutagenesis studies in human CRP (huCRP), we mutated Phe66 and Glu81 in the rabbit CRP (rbCRP) gene and generated a strain of transgenic mice (F66Y/E81K), which expressed this variant form of rbCRP. We also mutated Tyr175 in rbCRP to generate transgenic mice which expressed a variant form of rbCRP (Y175A). In vitro, F66Y/E81K rbCRP purified from serum had dramatically reduced binding to PCh. Additionally F66Y/E81K rbCRP not only maintained its ability to bind polycations and histones, but also bound more avidly to specific histones and lysine polymers than wild type (wt) rbCRP. Y175A rbCRP was not able to activate complement when bound to pneumococcal C-polysaccharide (PnC), but was, along with F66Y/E81K and wild type rbCRP, able to activate complement when bound to a small lysine polymer or when directly adsorbed to a solid phase. This complement activation presumably occurs through the classical complement pathway as the three rbCRPs, adsorbed to a solid phase, bound C1q. Taken together, our results demonstrate that the PCh-binding and the polycation-binding sites on rbCRP are distinct but possibly overlapping. The conformational changes in the C1q-binding site of CRP to activate complement depend on the nature of the ligand and on the location of the ligand-binding site.

Amino Acid Substitution↗
PubMed2003

Transcription factor c-Rel enhances C-reactive protein expression by facilitating the binding of C/EBPbeta to the promoter.

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPbeta, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus kappaB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a kappaB site diminished binding of C/EBPbeta to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPbeta-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus kappaB site did not abolish binding of C/EBPbeta to its binding site, indicating that this site was not necessary for the formation of C/EBPbeta-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPbeta-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus kappaB site. This finding indicates that Rel proteins are involved in the binding of C/EBPbeta to its binding site by a kappaB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPbeta-complexes and that c-Rel bound directly to C/EBPbeta-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPbeta. We conclude that c-Rel regulates CRP expression without the requirement of binding to a kappaB site, and binds directly to C/EBPbeta to facilitate the binding of C/EBPbeta to the CRP promoter.

Animals↗
PubMed2003

Overexpressed nuclear factor-kappaB can participate in endogenous C-reactive protein induction, and enhances the effects of C/EBPbeta and signal transducer and activator of transcription-3.

C-reactive protein (CRP), the prototypical human acute phase protein, is produced primarily by hepatocytes. Its expression is modestly induced by interleukin (IL)-6 in Hep3B cells while IL-1, which alone has no effect, synergistically enhances the effects of IL-6. In previous studies of the proximal CRP promoter, we found that signal transducer and activator of transcription-3 (STAT3) and C/EBPbeta -mediated IL-6-induced transcription and that Rel p50 acted synergistically with C/EBPbeta, in the absence of p65, to enhance CRP transcription. Neither a requirement nor a binding site for the classic nuclear factor (NF)-kappaB heterodimer p50/p65 were found. The current studies were undertaken to determine whether similar novel transcription factor interactions might regulate the endogenous CRP gene. Transiently overexpressed p50 or p65 induced CRP mRNA accumulation in Hep3B cells. The heterodimer p50/p65 was markedly more effective than p50 or p65 homodimers. Co-overexpression of p50 or p65 with C/EBPbeta or STAT3 synergistically enhanced CRP expression. Maximal expression was observed with overexpression of all four transcription factors; comparable effects were observed with IL-1beta treatment of cells overexpressing STAT3 + C/EBPbeta. Data from the Human Genome Project revealed 13 potential kappaB sites in the first 4000 bases of the CRP promoter, only one of which, centred at -2652, bound nuclear p50/p65 heterodimer activated by IL-1beta. Our findings indicate that classical NF-kappaB activation can participate in endogenous CRP induction, and that activated NF-kappaB may synergistically enhance the effects of C/EBPbeta and STAT3. They raise the possibility, not as yet established, that NF-kappaB activation may be responsible for the synergistic effect of IL-1beta on IL-6-induced CRP expression.

C-Reactive Protein↗
PubMed2003

A randomized prospective trial to assess the role of saline hydration on the development of contrast nephrotoxicity.

Though simple and attractive, the role of hydration for the prophylaxis of contrast nephrotoxicity has not been definitively established. We prospectively evaluated the role of deliberate saline hydration in patients undergoing nonemergency cardiac catheterization. Patients (n = 53) were randomized on the day prior to scheduled catheterization to one of two groups - group 1 (n = 27) received normal saline for 24 h (at a rate of 1 ml/kg/h) beginning 12 h prior to scheduled catheterization, and group 2 (n = 26) were allowed unrestricted oral fluids. Serum creatinine measured 24 and 48 h postcardiac catheterization was compared to the pre-randomization baseline value. The mean baseline calculated creatinine clearance was 79.6 +/- 31.9 ml/min and the mean baseline creatinine was 106 +/- 28 micromol/l. An increase in serum creatinine by at least 44.2 micromol/l (0.5 mg/dl), within 48 h of contrast exposure, was considered to represent clinically significant acute renal insufficiency. Ten subjects (18.9%) developed acute renal insufficiency. The incidence of acute renal insufficiency was significantly lower in group 1 (1 out of 27) as compared to group 2 (9 out of 26; p = 0.005 for comparison between groups; relative risk 0.11, 95% confidence interval 0.015 to 0.79). Twenty-four hours after contrast exposure, the mean increase in creatinine was less in group 1 vs. group 2 (8 +/- 11 vs. 20 +/- 21 micromol/l, p = 0.02). The increase in creatinine was not significantly different in group 1 vs. group 2 48 h after contrast exposure (12 +/- 21 vs. 29 +/- 40 micromol/l, p = 0.17). Deliberate saline hydration decreases the incidence of contrast-related acute renal failure and the severity of contrast-induced renal dysfunction in patients undergoing non-emergency cardiac catheterization.

Aged↗
PubMed2002

A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide.

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.

Amino Acid Substitution↗