Biomedical subjects

A J Szalai

Publications and source records attributed to A J Szalai.

At least 19 recordsLinked to original sources

PubMed2005

No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E*3-leiden/human C-reactive protein transgenic mice.

OBJECTIVE: C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. METHODS AND RESULTS: Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2+/-6.5 mg/L in male E3L/CRP mice, 0.2+/-0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. CONCLUSIONS: This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

Animals↗

PubMed2005

Systemic lupus erythematosus in a multiethnic US Cohort (LUMINA). XXX: association between C-reactive protein (CRP) gene polymorphisms and vascular events.

OBJECTIVES: To determine if a polymorphic GTn repeat in the intron of the C-reactive protein (CRP) gene associates with occurrence of vascular arterial events in systemic lupus erythematosus (SLE). METHODS: We performed a nested case-control study on the LUMINA cohort of 546 Hispanic, African-American and Caucasian SLE patients. Twenty-five patients who developed vascular arterial events (i.e. myocardial infarction, angina, coronary artery bypass graft surgery, stroke, claudication, gangrene or significant tissue loss and/or arterial peripheral thrombosis) after enrolment were selected as cases and 32 ethnically matched patients with no previous vascular arterial events served as controls. Their CRP gene GTn polymorphism and plasma CRP was determined. RESULTS: Patients with vascular events had more severe SLE and were more likely to have plasma CRP in the highest quintile of measured values. The overall distribution of GTn alleles for patients with vascular events had a greater number of the GT20 variant compared with controls [26.0% of alleles (13/50) vs 15.6% (10/64)]. This greater number of GT20 in patients with vascular events was observed for African-Americans [29.2% (7/24) vs 21.0% (8/38)] and Hispanics [33.0% (4/12) vs 0% (0/16)] but not for Caucasians [14.3% (2/14) vs 20.0% (2/10)]. For African-Americans and Hispanics combined (45 patients), the frequency of GT20 in those with vascular events (30.6%, 11/36) was significantly higher than in those without them (14.8%, 8/54) (P<0.05, one-tailed test for difference in proportions). When patients were categorized according to the number of GT20 alleles they carried (thus GT20/GT20, GT20/GTx or GTx/GTx, where x is any allele other than GT20), for both African-Americans and Hispanics the likelihood of vascular arterial events increased in proportion with the GT20 dose, and all GT20-homozygous patients developed vascular arterial events. CONCLUSIONS: The CRP GT20 variant is more likely to occur in African-American and Hispanic SLE patients than in Caucasian ones, and SLE patients carrying the GT20 allele are more likely to develop vascular arterial events.

Adult↗

PubMed2005

Single-nucleotide polymorphisms in the C-reactive protein (CRP) gene promoter that affect transcription factor binding, alter transcriptional activity, and associate with differences in baseline serum CRP level.

To investigate whether functional polymorphisms exist in the C-reactive protein (CRP) gene, i.e., ones that contribute directly to differences in baseline CRP among individuals, we sequenced a 1,156-nucleotide-long stretch of the CRP gene promoter in 287 ostensibly healthy people. We identified two single-nucleotide polymorphisms (SNPs), a bi-allelic one at nucleotide -409 (G-->A), and a tri-allelic one at -390 (C-->T-->A), both resident within the hexameric core of transcription factor binding E-box elements. Electrophoretic mobility shift assays confirmed that the SNP within the sequence (-412)CACGTG(-407) (E-box 1) modulates transcription factor binding, and that the one within (-394)CACTTG(-389) (E-box 2) supports transcription factor binding only when the -390 T allele is present. The commonest of four E-box 1/E-box 2 haplotypes (-409G/-390T) identified in the population supported highest promoter activity in luciferase reporter assays, and the rarest one (-409A/-390T) supported the least. Importantly, serum CRP in people with these haplotypes reproduced this rank order, i.e., people with the -409G/-390T haplotype had the highest baseline serum CRP (mean +/- SEM 10.9 +/- 2.25 microg/ml) and people with the -409A/-390T haplotype had the lowest (5.01 +/- 1.56 microg/ml). Furthermore, haplotype-associated differences in baseline CRP were not due to differences in age, sex, or race, and were still apparent in people with no history of smoking. At least two other SNPs in the CRP promoter lie within E-box elements (-198 C-->T, E-box 4, and -861 T-->C, E-box 3), indicating that not only is the quality of E-box sites in CRP a major determinant of baseline CRP level, but also that the number of E-boxes may be important. These data confirm that the CRP promoter does encode functional polymorphisms, which should be considered when baseline CRP is being used as an indicator of clinical outcome. Ultimately, development of genetic tests to screen for CRP expression variants could allow categorization of healthy people into groups at high versus low future risk of inflammatory disease.

Adult↗

PubMed2004

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus.

The effect of complement on transgene expression was evaluated in vivo and in vitro using mice lacking complement components. Complement component 3 (C3) deficient mice (C3-/-) and appropriate wild-type controls were intravenously injected with a replication incompetent, luciferase-expressing normal Ad5 (Ad5Luc1), or fibritin-fiber Ad5 (Ad5FFLuc1). Repeated, noninvasive bioluminescence imaging was conducted over 35 days. Our data show for the first time that C3 facilitates both short- and long-term hepatic expression of luciferase following systemic delivery. C3-/- mice showed significantly less (P < 0.05) luciferase expression in their liver than treatment-matched wild-type mice when 2.3 x 10(9) (Ad5Luc1) and 4.0 x 10(9) (Ad5Luc1 or Ad5FFLuc1) viral particles (v.p.) were infused. The maximal difference in luciferase activity between C3-/- and wild-type mice was 99-fold difference at 3 days for the 2.3 x 10(9) v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5FFLuc1). Preincubation of Ad5Luc1 with wild-type, C1q-/-, or factor B (FB) deficient mouse sera for 5 min significantly (P < 0.05) increased transduction of mouse liver cells, as compared to preincubation with C3-/- sera or PBS. These results suggest the classical or alternate complement pathway enhances Ad5-mediated liver transduction.

Adenoviridae↗

PubMed2002

Association between baseline levels of C-reactive protein (CRP) and a dinucleotide repeat polymorphism in the intron of the CRP gene.

Elevation of baseline C-reactive protein (CRP) is associated with increased risk of cardiac disease. This increase might reflect low-grade inflammation, but differences in CRP serum levels might also have a genetic component. To test this possibility, we investigated whether a polymorphic GT-repeat in the intron of the CRP gene contributes to variation in baseline CRP. We found that the polymorphism was associated with differences in baseline CRP in both normal individuals and in patients with the inflammatory disease systemic lupus erythematosus, viz. donors carrying two GT(16) alleles, two GT(21)alleles, or GT(16/21) heterozygotes had two-fold lower serum CRP than those with other genotypes. The frequency of GT(16) and GT(21) was two-fold higher in Caucasians than in African-Americans, but there was no difference in allele distribution between patients and controls. It is not yet known how this genetic polymorphism mediates its effect on CRP expression, and it probably is not a systemic lupus erythematosus susceptibility factor. Rather, the CRP intron polymorphism likely modifies the disease phenotype. On the other hand, the fact that baseline CRP does have a genetic component suggests that in coronary disease, stratification of risk assessment based on CRP levels might be enhanced by consideration of this polymorphism.

Adult↗

PubMed2000

Attenuation of experimental autoimmune demyelination in complement-deficient mice.

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement's contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3(-/-) and factor B(-/-) mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3(-/-) and factor B(-/-) mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.

Animals↗

PubMed2000

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.

Acute-Phase Reaction↗

PubMed2000

The Arthus reaction in rodents: species-specific requirement of complement.

We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.

Animals↗

PubMed2000

Human C-reactive protein is protective against fatal Salmonella enterica serovar typhimurium infection in transgenic mice.

C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection by Streptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection with Salmonella enterica serovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulent Salmonella strain, the serum titer of anti-Salmonella immunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.

Animals↗

PubMed1999

C-reactive protein: a physiological activator of interleukin 6 receptor shedding.

The soluble interleukin 6 receptor (sIL-6R) circulates at elevated levels in various diseases. This suggests that inflammatory mediators control sIL-6R release. Through examination of human neutrophils, it was found that the acute phase reactant C-reactive protein (CRP) activates a threefold increase in sIL-6R production. Maximal release occurred after 30-60 min exposure to CRP (50 micrograms/ml), and was mimicked by peptides corresponding to amino acid residues 174- 185 and 201-206 of native CRP. A third peptide fragment (77-82) had no effect. Differential mRNA splicing did not account for the CRP-mediated release of sIL-6R, since this isoform was not detected in conditioned media. Furthermore, stimulation of neutrophils with CRP or with peptides 174-185 or 201-206 promoted a loss of membrane-bound IL-6R, suggesting release by proteolytic shedding. The metalloprotease inhibitor TAPI had only a marginal effect on CRP-mediated sIL-6R release, suggesting that shedding occurs via a mechanism distinct from that previously reported. It well established that IL-6 stimulates the acute phase expression of CRP. Our current findings demonstrate a novel relationship between these two mediators, since CRP may affect IL-6-mediated inflammatory events by enabling formation of the sIL-6R/IL-6 complex.

Acute-Phase Reaction↗

PubMed1999

Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.

Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.

Animals↗

PubMed1999

Tumor necrosis factor alpha receptor I is important for survival from Streptococcus pneumoniae infections.

Tumor necrosis factor alpha (TNF-alpha) is important in resistance to various microorganisms and provides signals to the target cells through two different receptors, TNF-alpha receptor I (TNFRI) (p55 receptor) and TNFRII (p75 receptor). To delineate the significance of the two different signaling pathways in resisting infections with extracellular bacteria, we examined the resistance of mice to Streptococcus pneumoniae (serotype 6B). TNF-alpha needs to be present early in infections, since one injection of wild-type mice with anti-TNF-alpha leads to an increased susceptibility of these mice to S. pneumoniae. TNF-alpha signaling through the p55 receptor (but not the p75 receptor) is crucial in resisting S. pneumoniae infections, because intraperitoneal injection of 100 CFU/mouse killed p55-deficient mice by day 2 of infection, whereas 1,000,000 CFU/mouse was needed to kill half of the control mice. p55-deficient mice do not show evidence of a deficient acute-phase response. All three types of mice (p55 deficient, p75 deficient, and normal) showed comparable rises in the levels of two acute-phase proteins (serum amyloid P and C3) at 24, 48, and 72 h after the experimental infections, and all of the mice showed comparable influxes of neutrophils to the site of infection. Finally, it was demonstrated that p55-deficient mice can be protected from the lethal effects of S. pneumoniae infection by injection of antibodies specific for S. pneumoniae polysaccharide capsule.

Acute-Phase Reaction↗

PubMed1999

Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the alpha chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

Animals↗

PubMed1999

C-reactive protein: structural biology and host defense function.

Human C-reactive protein is a Ca2+-binding acute phase-protein with binding specificity for phosphocholine. Recent crystallographic and mutagenesis studies have provided a solid understanding of the structural biology of the protein, while experiments using transgenic mice have confirmed its host-defense function. The protein consists of five identical protomers in cyclic symmetry. On one face of each protomer there is a binding site for phosphocholine consisting of two Ca2+ ions that ligate the phosphate group and a hydrophobic pocket that accommodates the methyl groups of phosphocholine. On the opposite face is a deep cleft formed by parts of the N and C termini and bordered by an alpha-helix. Mutational studies indicate that the C1q-binding site of the molecule is located at the open end of this cleft with Asp112 and Tyr175 representing contact residues. Using human C-reactive protein transgenic mice, we investigated the host defense functions of the protein. Transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice. This was attributable to an up to 400-fold reduction in bacteremia mediated mainly by the interaction of C-reactive protein with complement. A complement-independent host protective effect was also demonstrated.

Animals↗

PubMed1998

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice.

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.

Age Factors↗

PubMed1997

C-reactive protein: structural biology, gene expression, and host defense function.

Over the past few years substantial insight was gained into the biology and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected with Streptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.

Acute-Phase Reaction↗

PubMed1996

Role of complement in C-reactive-protein-mediated protection of mice from Streptococcus pneumoniae.

Expression of a human C-reactive protein (CRP) transgene has been shown to protect mice from lethal Streptococcus pneumoniae infection. In the present study, we used cobra venom factor-induced decomplementation to investigate the role of complement in this CRP-mediated protection. An intact complement system significantly reduced pneumococcal bacteremia at 24 h postinfection and extended median survival time of both CRP-transgenic and nontransgenic mice. However, mortality was significantly lowered only for CRP transgenic mice. The transgene significantly reduced bacteremia for both normocomplementemic and decomplemented mice, but it resulted in a significantly longer median survival time and lower mortality only for normocomplementemic mice. These data suggest that in vivo complement and CRP amplify each other's protective capacity, particularly during the early course of infection.

Animals↗

PubMed1995

Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice.

C-reactive protein (CRP) is an acute phase protein with a well known association with infection and other inflammatory conditions. Studies with use of purified CRP in in vitro assays provided early evidence that CRP has antibacterial activity. Subsequently it was shown that passively administered human CRP can protect mice from lethal infection with Streptococcus pneumoniae. In this study, we extend these observations to an in vivo model of host resistance by using human CRP transgenic mice. CRP transgenic mice experimentally infected with S. pneumoniae lived longer and had significantly lower mortality than their nontransgenic littermates. This increased resistance to infection was associated with q 10- to 400-fold reduction of bacteremia. Furthermore, male transgenics exhibited longer survival time than females, and this difference could be attributed to increased expression of CRP by males, which was mediated by testosterone. This study provides the first unequivocal evidence that CRP plays an important role in vivo in host defense against pneumococcal infections, and shows that sex hormones can affect expression of the human CRP transgene in mice.

Animals↗