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1.  Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans▿  
Journal of Bacteriology  2010;192(18):4669-4679.
The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the −10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.
PMCID: PMC2937423  PMID: 20639331
2.  Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production 
The two-component regulatory system (TCS) CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH TCS.
PMCID: PMC4295557  PMID: 25642214
Streptococcus pneumoniae; two-component regulatory system CiaRH; acetyl phosphate; alternative phosphorylation; pyruvate oxidase; acetate kinase
3.  The Streptococcus pneumoniae cia Regulon: CiaR Target Sites and Transcription Profile Analysis 
Journal of Bacteriology  2003;185(1):60-70.
The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.
PMCID: PMC141814  PMID: 12486041
4.  Multilevel Control of Competence Development and Stress Tolerance in Streptococcus mutans UA159  
Infection and Immunity  2006;74(3):1631-1642.
Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.
PMCID: PMC1418624  PMID: 16495534
5.  Pneumococcal HtrA Protease Mediates Inhibition of Competence by the CiaRH Two-Component Signaling System 
Journal of Bacteriology  2005;187(12):3969-3979.
Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.
PMCID: PMC1151733  PMID: 15937159
6.  Identification of genes for small non-coding RNAs that belong to the regulon of the two-component regulatory system CiaRH in Streptococcus 
BMC Genomics  2010;11:661.
Post-transcriptional regulation by small RNAs (sRNAs) in bacteria is now recognized as a wide-spread regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH two-component regulatory system. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects β-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent regulatory events will be the key to define the physiological role of this important control system.
Genes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision.
The results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of the regulon of the two-component regulatory system CiaRH in all streptococci.
PMCID: PMC3091779  PMID: 21106082
7.  The CiaRH System of Streptococcus pneumoniae Prevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations in pbp2x Involved in β-Lactam Resistance 
Journal of Bacteriology  2006;188(5):1959-1968.
The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.
PMCID: PMC1426552  PMID: 16484208
8.  The CiaR Response Regulator in Group B Streptococcus Promotes Intracellular Survival and Resistance to Innate Immune Defenses ▿  
Journal of Bacteriology  2008;191(7):2023-2032.
Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS ΔciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and ΔciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.
PMCID: PMC2655536  PMID: 19114476
9.  Multiple Two-Component Systems Modulate Alkali Generation in Streptococcus gordonii in Response to Environmental Stresses▿  
Journal of Bacteriology  2009;191(23):7353-7362.
The oral commensal Streptococcus gordonii must adapt to constantly fluctuating and often hostile environmental conditions to persist in the oral cavity. The arginine deiminase system (ADS) of S. gordonii enables cells to produce, ornithine, ammonia, CO2, and ATP from arginine hydrolysis, augmenting the acid tolerance of the organism. The ADS genes are substrate inducible and sensitive to catabolite repression, mediated through ArcR and CcpA, respectively, but the system also requires low pH and anaerobic conditions for optimal activation. Here, we demonstrate that the CiaRH and ComDE two-component systems (TCS) are required for low-pH-dependent expression of ADS genes in S. gordonii. Further, the VicRK TCS is required for optimal ADS gene expression under anaerobic conditions and enhances the sensitivity of the operon to repression by oxygen. The known anaerobic activator of the ADS, Fnr-like protein (Flp), appeared to act independently of the Vic TCS. Mutants of S. gordonii lacking components of the CiaRH, ComDE, or VicRK grew more slowly in acidified media and were more sensitive to killing at lethal pH values and to agents that induce oxidative stress. This study provides the first evidence that TCS can regulate the ADS of bacteria in response to specific environmental signals and reveals some notable differences in the contribution of CiaRH, ComDE, and VicRK to viability and stress tolerance between the oral commensal S. gordonii and the oral pathogen Streptococcus mutans.
PMCID: PMC2786566  PMID: 19783634
10.  Relevance of the two-component sensor protein CiaH to acid and oxidative stress responses in Streptococcus pyogenes 
BMC Research Notes  2014;7:189.
The production of virulence proteins depends on environmental factors, and two-component regulatory systems are involved in sensing these factors. We previously established knockout strains in all suspected two-component regulatory sensor proteins of the emm1 clinical strain of S. pyogenes and examined their relevance to acid stimuli in a natural atmosphere. In the present study, their relevance to acid stimuli was re-examined in an atmosphere containing 5% CO2.
The spy1236 (which is identical to ciaHpy) sensor knockout strain showed significant growth reduction compared with the parental strain in broth at pH 6.0, suggesting that the Spy1236 (CiaHpy) two-component sensor protein is involved in acid response of S. pyogenes. CiaH is also conserved in Streptococcus pneumoniae, and it has been reported that deletion of the gene for its cognate response regulator (ciaRpn) made the pneumococcal strains more sensitive to oxidative stress. In this report, we show that the spy1236 knockout mutant of S. pyogenes is more sensitive to oxidative stress than the parental strain.
These results suggest that the two-component sensor protein CiaH is involved in stress responses in S. pyogenes.
PMCID: PMC3986815  PMID: 24673808
11.  The Campylobacter jejuni CiaC virulence protein is secreted from the flagellum and delivered to the cytosol of host cells 
Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute C. jejuni-mediated disease (campylobacteriosis) involves C. jejuni invasion of host epithelial cells using adhesins (e.g., CadF and FlpA) and secreted proteins [e.g., the Campylobacter invasion antigens (Cia)]. The genes encoding the Cia proteins are up-regulated upon co-culture of C. jejuni with epithelial cells. One of the Cia proteins, CiaC, is required for maximal invasion of host cells by C. jejuni. Previous work has also revealed that CiaC is, in part, responsible for host cell cytoskeletal rearrangements that result in membrane ruffling. This study was performed to test the hypothesis that CiaC is delivered to the cytosol of host cells. To detect the delivery of CiaC into cultured epithelial cells, we used the adenylate cyclase domain (ACD) of Bordetella pertussis CyaA as a reporter. In this study, we found that export and delivery of the C. jejuni Cia proteins into human INT 407 epithelial cells required a functional flagellar hook complex composed of FlgE, FlgK, and FlgL. Assays performed with bacterial culture supernatants supported the hypothesis that CiaC delivery requires bacteria-host cell contact. We also found that CiaC was delivered to host cells by cell-associated (bound) bacteria, as judged by experiments performed with inhibitors that specifically target the cell signaling pathways utilized by C. jejuni for cell invasion. Interestingly, the C. jejuni flgL mutant, which is incapable of exporting and delivering the Cia proteins, did not induce INT 407 cell membrane ruffles. Complementation of the flgL mutant with plasmid-encoded flgL restored the motility and membrane ruffling. These data support the hypothesis that the C. jejuni Cia proteins, which are exported from the flagellum, are delivered to the cytosol of host cells.
PMCID: PMC3417660  PMID: 22919623
T3SS; flagellum; effector proteins; adenylate cyclase; membrane ruffling
12.  Bacteriocin Activity of Streptococcus pneumoniae Is Controlled by the Serine Protease HtrA via Posttranscriptional Regulation▿  
Journal of Bacteriology  2008;191(5):1509-1518.
The blp locus of a type 6A strain of Streptococcus pneumoniae encodes a two-peptide bacteriocin, pneumocin MN, which mediates intraspecies competition during mouse nasopharyngeal colonization. This locus is regulated by a quorum-sensing mechanism consisting of a dedicated two-component regulatory system and a peptide pheromone. Like most clinical isolates, this type 6A strain can be separated into opaque and transparent colony variants, each playing a different role during pneumococcal infection. In this study, we show that the blp locus is differentially regulated at the posttranscriptional level in pneumococcal opacity variants. Transparent and opaque variants produce equivalent amounts of blpMNPO transcript when stimulated with a synthetic pheromone, but transparent variants have no pneumocin MN-mediated inhibitory activity while opaque variants produce large zones of inhibitory activity. The differential regulation in opacity variants is driven by the two-component regulatory system CiaRH via its regulation of the serine protease HtrA. Transparent mutants deficient in CiaH or HtrA show increased pneumocin MN-mediated inhibition. In addition, these mutants demonstrate alterations in their dose response to a synthetic peptide pheromone, suggesting that HtrA activity impacts pneumocin MN production at the level of signaling. This, in addition to its known effects on competence, suggests that HtrA is a pleiotropic regulator whose protease activity affects several important bacterial pathways. The complex regulation of pneumocins may allow the pneumococcus to reserve the secretion of active peptides for situations where the benefit of their inhibitory activity outweighs the cost of their production.
PMCID: PMC2648213  PMID: 19103930
13.  Human CIA2A (FAM96A) and CIA2B (FAM96B) integrate maturation of different subsets of cytosolic-nuclear iron-sulfur proteins and iron homeostasis 
Cell metabolism  2013;18(2):187-198.
Numerous cytosolic and nuclear proteins involved in metabolism, DNA maintenance, protein translation, or iron homeostasis depend on iron-sulfur (Fe/S) cofactors, yet their assembly is poorly defined. Here, we identify and characterize human CIA2A (FAM96A), CIA2B (FAM96B), and CIA1 (CIAO1) as components of the cytosolic Fe/S protein assembly (CIA) machinery. CIA1 associates with either CIA2A or CIA2B and the CIA targeting factor MMS19. The CIA2B-CIA1-MMS19 complex binds to and facilitates assembly of most cytosolic-nuclear Fe/S proteins. In contrast, CIA2A specifically matures iron regulatory protein (IRP) 1 which is critical for cellular iron homeostasis. Surprisingly, a second layer of iron regulation involves the stabilization of IRP2 by CIA2A binding or upon depletion of CIA2B or MMS19, even though IRP2 lacks a Fe/S cluster. In summary, CIA2B-CIA1-MMS19 and CIA2A-CIA1 assist different branches of Fe/S protein assembly, and intimately link this process to cellular iron regulation via IRP1 Fe/S cluster maturation and IRP2 stabilization.
PMCID: PMC3784990  PMID: 23891004
14.  Streptococcus pneumoniae Biofilm Formation Is Strain Dependent, Multifactorial, and Associated with Reduced Invasiveness and Immunoreactivity during Colonization 
mBio  2013;4(5):e00745-13.
Biofilms are thought to play an important role during colonization of the nasopharynx by Streptococcus pneumoniae, yet how they form in vivo and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from in vitro biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust in vitro biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with ΔpsrP mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells in vitro despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response.
This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.
PMCID: PMC3812715  PMID: 24129258
15.  Control of Virulence by the Two-Component System CiaR/H Is Mediated via HtrA, a Major Virulence Factor of Streptococcus pneumoniae 
Journal of Bacteriology  2004;186(16):5258-5266.
The CiaR/H two-component system is involved in regulating virulence and competence in Streptococcus pneumoniae. The system is known to regulate many genes, including that for high-temperature requirement A (HtrA). This gene has been implicated in the ability of the pneumococcus to colonize the nasopharynx of infant rats. We reported previously that deletion of the gene for HtrA made the pneumococcal strains much less virulent in mouse models, less able to grow at higher temperatures, and more sensitive to oxidative stress. In this report, we show that the growth phenotype as well as sensitivity to oxidative stress of ΔciaR mutant was very similar to that of a ΔhtrA mutant and that the expression of the HtrA protein was reduced in a ciaR-null mutant. Both the in vitro phenotype and the reduced virulence of ΔciaR mutant could be restored by increasing the expression of HtrA.
PMCID: PMC490881  PMID: 15292127
16.  Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis 
Arthritis Research  1999;1(1):81-91.
Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.
Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-α seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockade of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-4 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+ T cells and it promotes the maturation of Th2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-α, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.
Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.
Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.
DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.
Treatment of established CIA with IL-4 (1 μg/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 μg/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.
In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.
Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.
Effects in mice of treatment with interleukin-4 or control on disease activity, cartilage damage and bone destruction. Mice were treated intraperitoneally for 7 days with either vehicle (control) or 1 μg/day interleukin-4 (IL-4). CIA, collagen-induced arthritis. *P < 0.05, versus control, by Mann-Whitney U test.
PMCID: PMC17779  PMID: 11056663
bone destruction; cartilage oligomeric matrix protein levels; collagen-induced arthritis; interleukin-4; prednisolone
17.  Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties 
Letters in applied microbiology  2007;45(4):398-404.
To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties.
Methods and Results
For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development.
Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis.
Significance and Impact of the Study
Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.
PMCID: PMC2062497  PMID: 17897382
acid resistance; biofilm formation; gene inactivation; oral streptococci; stress tolerance; two-component system
18.  Cia5d regulates a new fibroblast-like synoviocyte invasion-associated gene expression signature 
The in vitro invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) have been shown to correlate with disease severity and radiographic damage. We recently determined that FLSs obtained from pristane-induced arthritis (PIA)-susceptible DA rats are also highly invasive in the same in vitro assay through Matrigel. The transfer of alleles derived from the arthritis-resistant F344 strain at the arthritis severity locus Cia5d (RNO10), as in DA.F344(Cia5d) congenics, was enough to significantly and specifically reduce the invasive properties of FLSs. This genetically controlled difference in FLS invasion involves increased production of soluble membrane-type 1 matrix metalloproteinase (MMP) by DA, and is dependent on increased activation of MMP-2. In the present study we aimed to characterize the pattern of gene expression that correlates with differences in invasion in order to identify pathways regulated by the Cia5d locus.
Synovial tissues were collected from DA and DA.F344(Cia5d) rats 21 days after the induction of PIA. Tissues were digested and FLSs isolated. After a minimum of four passages, FLSs were plated on Matrigel-covered dishes at similar densities, followed by RNA extraction. Illumina RatRef-12 expression BeadChip arrays were used. Expression data were normalized, followed by t-test, logistic regression, and cluster analysis. Real-time PCR was used to validate the microarray data.
Out of the 22,523 RefSeq gene probes present in the array, 7,665 genes were expressed by the FLSs. The expression of 66 genes was significantly different between the DA and DA.F344(Cia5d) FLSs (P < 0.01). Nineteen of the 66 differentially expressed genes (28.7%) are involved in the regulation of cell cycle progression or cancer-associated phenotypes, such as invasion and contact inhibition. These included Cxcl10, Vil2 and Nras, three genes that are upregulated in DA and known to regulate MMP-2 expression and activation. Nine of the 66 genes (13.6%) are involved in the regulation of estrogen receptor signaling or transcription. Five candidate genes located within the Cia5d interval were also differentially expressed.
We have identified a novel FLS invasion associated gene expression signature that is regulated by Cia5d. Many of the genes found to be differentially expressed were previously implicated in cancer cell phenotypes, including invasion. This suggests a parallel in the behavior of arthritis FLSs and cancer cells, and identifies novel pathways and genes for therapeutic intervention and prognostication.
PMCID: PMC2575606  PMID: 18706093
19.  Deletion of Fcγ Receptor IIB Renders H-2b Mice Susceptible to Collagen-induced Arthritis  
Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcγRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcγRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2b haplotype. H-2b mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcγRIIB-deficient, H-2b mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcγRIIB-deficient mice developing CIA (6.9 ± 3.6) was comparable to that of DBA/1 mice (8.6 ± 1.9), an H-2q strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcγRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcγRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2b haplotype can be rendered permissive to CIA induction through deletion of FcγRIIB, suggesting that FcγRIIB plays a critical role in suppressing the induction of CIA.
PMCID: PMC1887699  PMID: 9874575
collagen-induced arthritis; autoimmunity; Fc receptor; gene targeting; macrophage
20.  Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis 
Arthritis Research  2000;2(6):504-512.
We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.
Accumulation of fibrin in the joints remains one of the most striking histopathological features of rheumatoid arthritis (RA). Recently, we have provided evidence of the deleterious role of synovial fibrin deposition in arthritic joints in antigen-induced arthritis (AIA), a well-established murine model of RA.
A local imbalance between fibrin formation and fibrin dissolution may result in fibrin deposition in the joints.
On the one hand, fibrin formation is mainly initiated by tissue factor (TF), a transmembrane protein serving as a receptor for factor VII. Under normal conditions, TF expression and activity are tightly regulated. Constitutive TF expression is restricted to perivascular and epithelial cells, and the catalytic activity of the TF/VIIa complex can be inhibited by tissue factor pathway inhibitor (TFPI). Pathological conditions can perturb the cell-type-restricted pattern of TF expression. In particular, recent reports have shown that transcriptional activation of TF can be mediated by molecular mechanisms involving induction of the early growth response gene 1 (EGR1) or of the protease-activated receptor (PAR1) or vascular endothelial growth factor (VEGF) genes.
On the other hand, fibrin degradation is mediated primarily by plasmin, which is the active form of the zymogen plasminogen. Conversion of plasminogen to plasmin is under the control of serine protease plasminogen activators, such as the urokinase plasminogen activator (uPA), and their inhibitors, such as the plasminogen activator inhibitor (PAI-1).
We hypothesized that the deposition of fibrin in the joints may result from an imbalance in the local expression of key genes involved in coagulation and fibrinolytic pathways. To test this hypothesis, we investigated mRNA levels in arthritic versus nonarthritic joint tissues from two murine models of RA: AIA and collagen-induced arthritis (CIA). Genes that are directly implicated in coagulation (TF, TFPI) and fibrinolysis (UPA, PAI1), and other genes that may influence the expression of TF (EGR1, PAR1, VEGF), were investigated using a novel multiprobe RNase protection assay (RPA). Furthermore, we evaluated coagulation activity in arthritic and nonarthritic mice.
Mice with AIA or CIA were sacrificed at different time points: 2, 4, and 16 h and 3, 7, and 14 d after intra-articular antigen injection for AIA; 42 d after the first immunization for CIA. Total RNA was prepared from arthritic and nonarthritic knees for AIA, or arthritic and nonarthritic hind paws for CIA. Messenger RNA (mRNA) levels of the genes described above were determined by RPA and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. Coagulation assays were performed on joint tissue extracts and concentrations of thrombin-antithrombin III (TAT) complex were measured in plasma.
In AIA, all the genes studied except VEGF were upmodulated as early as 2 h. PAR1, TFPI, EGR1, and UPA expression decreased to control levels by 16 h, whereas the expression of TF and PAI1 remained elevated. At later times, only TF, PAI1, and UPA showed sustained overexpression. In CIA, gene expression was assayed at only one time point (42 d after immunization) and all genes showed higher mRNA levels in the affected paws than in control paws. In AIA mice, procoagulant activity and TF activity were significantly increased in arthritic joints, and in CIA mice, plasma TAT levels were significantly enhanced.
Fibrin deposition in synovia is prominent in both RA and experimental arthritis, suggesting that this protein may play a role in the pathogenesis of chronic inflammation. In this study, we have tried to shed some light on the molecular mechanisms leading to extravascular fibrin deposition, using two well-established mouse models of RA: AIA and CIA. The kinetics of gene expression was first analyzed in mice with AIA, because this model allows for an accurate, temporally controlled sampling of synovial inflammation. We then extended our observations by analyzing one time point in CIA, 42 d after immunization, when chronic inflammation is present. We found that in both models, coagulation and fibrinolysis in arthritic joints were significantly increased, and that the most significant increases were in TF and PAI-1.
Although the molecular mechanism or mechanisms responsible for the transcriptional changes observed are not completely understood, the increases in TF, PAI-1, and uPA are probably due to the production of proinflammatory cytokines such as IL-1 and TGF-α. These cytokines, whose presence in the inflamed synovium is well documented, are known to induce these genes through the activation of nuclear factor κB (NF-κB), a transcription factor. TF induction is also under the control of a proximal enhancer containing a binding site for the inducible transcription factor EGR1. Indeed, the early rise of EGR1 expression in AIA is consistent with its classification as immediate-early gene and may be responsible for the induction of early expression of TF. Early TF stimulation in AIA can also be accounted for by the transient overexpression of PAR1. Contrary to what has been shown in RA, VEGF expression remained essentially unchanged throughout the progression of AIA, probably reflecting a peculiarity of this murine model.
The alteration of the patterns of gene expression was accompanied by increased functional coagulation activity, which was more marked in AIA than in CIA.
Prominent fibrin deposition in two different animal models of RA – AIA and CIA – can be attributed to modulations in key regulatory genes for coagulation and fibrinolysis.
PMCID: PMC17822  PMID: 11056680
arthritis; coagulation; fibrinolysis; mice; RNase protection
21.  Competence Modulation by the NADH Oxidase of Streptococcus pneumoniae Involves Signal Transduction 
Journal of Bacteriology  2001;183(2):768-772.
Oxygen controls competence development in Streptococcus pneumoniae. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by comC and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of ciaRH and comCDE. Thus, in S. pneumoniae, Nox, with O2 as its secondary substrate, is part of the O2-signaling pathway.
PMCID: PMC94936  PMID: 11133974
22.  Identification and Characterization of Noncoding Small RNAs in Streptococcus pneumoniae Serotype 2 Strain D39 ▿ †  
Journal of Bacteriology  2009;192(1):264-279.
We report a search for small RNAs (sRNAs) in the low-GC, Gram-positive human pathogen Streptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny, A. Brencic, S. Lory, and M. K. Waldor, Nucleic Acids Res. 34:3484-3493, 2006), we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann, M. Kovacs, R. Hakenbeck, and R. Bruckner, Mol. Microbiol. 66:110-126, 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [nt]; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ΔciaR mutants, but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR+ strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.
PMCID: PMC2798261  PMID: 19854910
23.  A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae 
Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.
PMCID: PMC4068597  PMID: 24777105
24.  Extracellular Matrix Formation Enhances the Ability of Streptococcus pneumoniae to Cause Invasive Disease 
PLoS ONE  2011;6(5):e19844.
During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease.
PMCID: PMC3097209  PMID: 21611130
25.  Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB 
Journal of Bacteriology  2001;183(15):4599-4608.
In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO4 in vitro. Thus, mutations which either reduced the stability of MicA-PO4 or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO4 is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.
PMCID: PMC95355  PMID: 11443095

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