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1 School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
2 Department of Pathology, Children's Hospital Los Angeles, CA 90027, USA
3 University of Southern California Keck School of Medicine Los Angeles, CA 90027, USA
4 Department of Neurology, Queen's Medical Centre NHS Trust, Nottingham NG7 2UH, UK
5 Bacterial Epidemiology and Antimicrobial Resistance Research Unit, USDA, Agricultural Research Service, 950 College Station Road, Athens, GA 30605, USA
Correspondence
Stacy M. Townsend
stacy.townsend{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Biering et al., 1989; Coignard et al., 2006; Himelright et al., 2002; Jarvis, 2005; Muytjens et al., 1983). This has generated significant concern over the non-sterile condition of this product and the regulatory standards governing the presence of this ubiquitous, opportunistic pathogen in infant formula powder (FAO-WHO, 2006). While recent studies have focused on the identification and classification of this genetically diverse species, very little is known concerning virulence (Drudy et al., 2006; Iversen et al., 2006; Iversen & Forsythe, 2007). Studies on toxin production by Pagotto et al. (2003) showed the production of cytotoxins or enterotoxins by clinical isolates and therefore these may contribute to a possible mechanism of pathogenesis. There is considerable diversity within the Ent. sakazakii species, which can be divided into four cluster groups according to 16S rDNA analysis (Iversen et al., 2004). It is of interest whether the virulence of Ent. sakazakii varies according to cluster group. The presence of lipopolysaccharide (LPS) in powdered infant formula has also been implicated in increasing the permeability of tissue barriers to intestinal bacteria including Ent. sakazakii (Townsend et al., 2007). Studies to determine the ability of Ent. sakazakii to penetrate the blood brain barrier (BBB) and cause meningitis are hindered due to lack of an animal model. Citrobacter spp. and Escherichia coli K1 elicit haematogenous meningitis and brain abscess development in the neonatal rat (Kim et al., 1997; Kline et al., 1988); however, similar dissemination is not observed with Ent. sakazakii and in vivo models of this infection may require the use of immunodeficient animal strains (Pagotto et al., 2007). This study utilized intracranial inoculation of neonatal rats with Ent. sakazakii to facilitate histological characterization of the inflammatory response in vivo. Ent. sakazakii strains from cluster groups 1 and 2 were shown to significantly invade rat brain capillary endothelial cells (rBCEC4). Gene probing suggests that Ent. sakazakii survival in human (U937) macrophages in vitro may be influenced by sodA. In addition, we suggest that Ent. sakazakii may bias early IL-10/IL-12 cytokine expression by macrophages, contributing to the exacerbation of disease.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The cluster 1 strain NTU658 (ATCC BAA-894) is currently being sequenced by Washington University in St Louis Genome Sequencing Center (http://www.genome.wustl.edu). Citrobacter koseri SMT319 (Townsend et al., 2003), Citrobacter freundii NTU340, E. coli K1 meningitic cerebrospinal fluid (CSF) isolate RS218 (O18 : K1 : H7) and Salmonella enterica serovar Enteritidis (NCTC 3046) were used as positive controls and non-pathogenic E. coli K-12 served as a negative control. For animal studies, gentamicin protection assays and cytokine secretion assays, bacteria were aerobically grown at 37 °C to mid-exponential phase in brain heart infusion (BHI) broth and washed twice with and resuspended in cold PBS. Concentrations of bacterial inoculations were determined by OD600 and confirmed by plate count enumeration.
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Superoxide dismutase (SOD) activity.
SOD activity in Ent. sakazakii cell lysates was evaluated using the Superoxide Dismutase Assay kit II (Calbiochem). Yersinia enterocolitica strain 8081 served as a positive control (Roggenkamp et al., 1997). Bacterial pellets collected from 2 ml overnight cultures were sonicated in ice-cold 20 mM HEPES buffer containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose (pH 7.2). Centrifugation at 1500 g for 5 min at 4 °C cleared the supernatants used in the assay to detect superoxide radicals according to the manufacturer's instructions. Two independent assays were performed in duplicate.
Tissue culture cell line cultivation and invasion assays.
U937 macrophages were obtained from the ATCC (CRL-1593.2) and seeded into 75 ml tissue culture flasks (Sundstrom & Nilsson, 1976). Cells were cultivated, activated and plated as described previously (Townsend et al., 2003). Cells were gently washed with RPMI to remove residual phorbol 12-myristate 13-acetate (PMA) following activation, and fresh medium was added prior to inoculation with unopsonized bacteria. U937 human macrophages were infected at an m.o.i. of 10 for 45 min at 37 °C in 5 % CO2. After a 45 min incubation period, the medium was aspirated and replaced with U937 macrophage medium supplemented with 100 µg gentamicin ml–1 and incubated for an additional 45 min at 37 °C in 5 % CO2. Macrophages were washed twice, lysed with 0.5 % Triton X, serially diluted, and plated to determine the number of intracellular bacteria at various time points (Zaidi et al., 1996). The viability of the bacterial strains tested was not affected by 0.5 % Triton X treatment. For persistence assays, cells were replenished daily with fresh medium containing 10 µg gentamicin ml–1 (above the MIC). Trypan Blue exclusion staining indicated that macrophage viability ranged from 80 to 95 % and was maintained for at least 96 h. For persistence assays, results for each time point are presented as the percentage of inoculum that was intracellular. All assays were performed in triplicate at least twice.
The rat brain capillary endothelial cell line (rBCEC4) was a kind gift from I. E. Blasig (Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany). Following the 22nd subculture rBCEC4 cells were seeded at 1x105 cells per well into collagen-coated 24-well plates and left to adhere for 48 h. The medium contained DMEM, 4.5 g glucose l–1, 1.2 mM glutamine, 100 U penicillin ml–1, 100 µg streptomycin ml–1, 2.5 µg amphotericin B ml–1 (Sigma-Aldrich), 100 µg heparin ml–1, 110 µg sodium pyruvate ml–1 (Sigma), 10 µg ECGF ml–1 (endothelial growth factor; Axxora), 10 % fetal bovine serum (Blasig et al., 2001). Each bacterial strain was grown in brain heart infusion broth (BHI; CM 10322, Oxoid) to mid-exponential phase from overnight cultures and inoculated in triplicate at an m.o.i. of 1 : 100. Inoculated cells were incubated with 5 % CO2 at 37 °C for 1.5 h as previously described (Badger et al., 1999). In order to quantify bacterial invasion 100 µg gentamicin ml–1 was added to each well and incubated for 30 min. Then the cells were washed twice with PBS and treated with trypsin to dislodge the adherent cells. The rBCEC4 cells were lysed with 0.5 % Triton X and serial dilutions were plated on nutrient agar. Cell integrity following invasion was qualitatively assessed using Trypan Blue staining after 2 h incubation. E. coli K-12 and C. koseri SMT319 were used as negative and positive controls, respectively. Data are presented as percentage invasion as determined by (Number of bacteria recovered/Number of bacteria inoculated)x100.
Animal studies.
Timed-pregnant (E14) Sprague–Dawley rats (Charles River Laboratories) were obtained and gave birth in our vivarium after a 21 day gestation period. Litters averaged 12 pups and were kept with their mother in an opaque polypropylene cage under a Small Animal Isolator (Forma Scientific). Two- to three-day-old rat pups were anaesthetized with isoflurane and inoculated. For serum cytokine studies (described below), 107 c.f.u. in 0.1 ml PBS were inoculated intraperitoneally (i.p.). For histological studies, 103 c.f.u. in 0.002 ml PBS were inoculated intracranially (i.c.); i.c. inoculations were administered through a burr hole produced by a 26-gauge needle at coordinates approximately 5 mm caudal to the right eye and 2 mm right of the sagittal suture. A 33-gauge, single internal cannula (Plastics One) was attached to a Hamilton 1801RN 10 µl syringe (Hamilton Company) with a 22-gauge needle via 24-gauge standard wall tubing, to administer the dose. The needle was inserted perpendicular into the right parietal area approximately 2 mm deep from the external surface; 2 µl was injected over 1 min then the needle was carefully retracted. For the study reported in Table 2, rat pups were anaesthetized as they succumbed to infection up to 9 days post i.c. inoculation. Blood samples were aseptically collected via intracardiac puncture, as previously described (Badger & Kim, 1998). Blood samples (10 µl) were inoculated in LB and plated on agar plates. Rats were euthanized and whole brains were removed for histological analysis. All animal experiments were performed according to protocols approved by CHLA Institutional Animal Care and Use Committee (IACUC).
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Electron microscopy.
Transmission electron microscopy was used to visualize Ent. sakazakii within rat macrophages. Ent. sakazakii-infected rat brain samples were fixed with 2.5 % glutaraldehyde in 0.1 M PBS. Samples were post-fixed for 1 h with 2 % OsO4, rinsed, dehydrated through graded ethanol solutions, and embedded in polypropylene oxide. Ultrathin sections mounted on collodion grids (single hole) were stained with uranyl acetate and lead citrate and examined with a Philips CM transmission electron microscope.
DNA isolation, gene identification and PCR.
Genomic DNA was prepared from 1.5 ml overnight culture grown in LB broth using a GenElute Bacterial Genomic DNA kit (Sigma) according to the manufacturer's instructions. DNA–DNA microarray hybridization of Ent. sakazakii DNA to Salmonella enterica serotype Typhi and Typhimurium virulence gene arrays (Porwollik et al., 2003) identified homologues to Ent. sakazakii genes sodA and ompA related to macrophage survival and serum resistance. The Ent. sakazakii genome project at Washington University School of Medicine Genome Sequencing Center (http://www.genome.wustl.edu) provided sequence information of the Ent. sakazakii gene homologous to sodA and sodB from S. Typhimurium and PCR primers were designed to probe the chromosomal DNA. The PCR reaction mixture contained 5x Green GoTaq Flexi Buffer (Promega), 2.5 mM MgCl2, 0.2 mM each dNTP, 0.5 µM SodA1-F (5'-GTCAACAACGCTAACG-3') and SodA1-R (5'-CCCATCAGCGGGGAAT-3') primers, 2.5 U GoTaq Flexi DNA polymerase (Promega), 100 ng template DNA, and nuclease-free water to a final volume of 50 µl. The thermocycler (Genius FGEN 05 TD; Techne) was programmed as follows. An initial denaturing step at 94 °C for 2 min was followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 1 min, and a final extension step of 72 °C for 5 min; this yielded a 352 bp product. The ompA gene has recently been identified and was amplified using primers ESSF and ESSR, which yield a 469 bp product following PCR conditions described previously (Mohan Nair & Venkitanarayanan, 2006). PCR products were visualized on 1 % agarose gels stained with 0.5 µg ethidium bromide ml–1.
Quantitative analysis of cytokine secretion.
ELISA was used to quantify cytokine secretion (IL-10, IL-12 and TNF
) from U937 macrophages following Ent. sakazakii uptake. Human IL-10 (sensitivity <1 pg IL-10 ml–1), IL-12 (sensitivity <0.2 pg IL-12 ml–1), and TNF (sensitivity <1.7 pg TNF ml–1) ELISA kits (Biosource International) were used to measure cytokines secreted into the macrophage supernatant at 6 and 24 h post-inoculation with each strain in this study. E. coli O111 : B4 LPS or PMA (Sigma) was used as? a positive control. ELISA was also used to measure rat pup IL-10 (sensitivity <5 pg IL-10 ml–1) concentrations (Biosource International). Rat serum was isolated 24 h after i.p. injection of Ent. sakazakii strain NTU2 or NTU658 and IL-10 concentration was measured. In accordance with the manufacturer's instructions, each sample (serum was diluted 1 : 10) was run in duplicate wells and the absorbance values were averaged. Standard curves were generated using the cytokine standards supplied by the manufacturer.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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For the comparative study summarized in Table 2
, strains from clusters 1 and 2 were inoculated into rat pups via intracranial injection. Blood and brains were collected and examined between 6 and 9 days post-inoculation to observe evidence of sepsis and chronic-pattern inflammation. This study showed that NTU1 was attenuated in comparison to other strains such as NTU2, which initiated chronic-pattern inflammation (lymphocytic predominance) in 83 % of rats tested (Table 2). This provides evidence that even within genetically similar strains there are significant differences in the host immune response. These differences may be attributed to distinctions at the level of protein expression, although it cannot be ruled out that they may be due to extensive subculturing resulting in a loss of virulence in NTU1. Strain NTU658, from cluster group 1, is reported as having caused a neonatal outbreak and its genome is currently being sequenced. This strain caused meningitis and chronic-pattern inflammation in 33 % of infected rat pups (Table 2). Strain NTU57 (cluster group 2) also caused chronic-pattern inflammation in 33 % of infected rat pups, and 50 % of the rat pups developed meningitis as defined by inflammation within the meninges following histological analysis (Table 2). No bacterial growth was detected in blood samples; thus no evidence of sepsis was obtained. This comparative study identified strain NTU2 as having the highest occurrence of chronic-pattern inflammation, and this strain was used for further kinetic studies.
Intracranial injection of Ent. sakazakii was performed in a kinetic study to describe the progression of brain inflammation. The injection site was located in the right cerebral cortex at the approximate junction of the forebrain and midbrain at a depth (2 mm) not penetrating the ventricle. Rat pups were sacrificed at various time points up to 9 days. Histological analysis of H&E-stained coronal sections elucidated the inflammatory response. Fig. 1(a) is an example of normal brain histology observed in rat pups 9 days post-infection with NTU1 (most indistinguishable from controls) to compare to the severe inflammatory reaction observed in NTU2-infected rat pups. Only three rat pups inoculated with NTU1 had extremely small foci of inflammatory cells (mostly neutrophils) in the cingular gyrus, near the base of the brain, or near the meninges. Rat pups inoculated with NTU2 had severe bilateral ventriculitis, meningitis and marginalized neutrophils in local blood vessels 3 days post-inoculation (Fig. 1b, c). Lymphocytes and microglia were activated, reactive astrocytes were observed and inflammation was associated with a micro-haemorrhage (Fig. 1d). The occurrence of oedema and flocculation suggests that increased vascular permeability and fibrin liberated from blood vessels may impair drainage of CSF, causing hydrocephalus. The choroid plexus also harboured inflammatory cells and secreted fibrin, which was not prevalent in the C. koseri model (Townsend et al., 2003). Leptomeningitis, ventriculitis and ventriculomegaly, with massive dilation of the ventricles, worsened 6 days post-inoculation (Fig. 1e). Morphological evidence of neuronal or glial cell death via apoptosis was suggested by hyperchromatic, condensed nuclei within the cortex (Fig. 1f) and could be induced by bacterial factors such as LPS or other secreted endotoxins (Pagotto et al., 2003). Further, neuronal death and cell lyses releases acid hydrolase and causes liquefaction and necrosis of brain tissue. It follows that there is also evidence of ischaemic damage (possibly due to oedema and pressure), reduced neuronal density and liquefaction within the cortex; these factors could also influence the observed neuronal cell death. Neutrophils were still marginated and infiltrating into the cortex with other inflammatory cells, causing multiple foci of cerebritis and small ischaemic lesions more than a week after inoculation (Fig. 1g, h, i).
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). Multiple inflammatory foci within the brain closely associated with blood vessels suggest that in this model bacteria travel down the perivascular spaces (Fig. 1). This suggests a way that the bacteria may enter the CSF, causing a massive influx of inflammatory cells into the ventricles and meninges that may break down adhesion junctions and give access to the brain parenchyma. Micro-abscesses, inflammatory foci and marginated neutrophils were visible at day 9 (Fig. 1k, l). Electron microscopy was used to further discern the host cell interactions and intracellular location of internalized bacteria. Electron microscopic analysis of infant rat brains 7 days post-inoculation found Ent. sakazakii in spacious membrane-bound phagosomes within infiltrating neutrophils and macrophages associated with inflammation (Fig. 2).
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). The C. koseri positive control was extremely invasive (2.95 %) while E. coli K-12 did not invade this cell line (Fig. 3). Strains NTU1, NTU3 and NTU84 were at least twofold less invasive than E. coli K1 (P
0.0002) in vitro (Fig. 3) and this has been shown to be biologically relevant since 0.1 % invasion is associated with enhanced CNS entry in vivo (Badger & Kim, 1998; Wang et al., 1999; Badger et al., 2000). Strains NTU1, NTU3 and NTU84 were also significantly less invasive than NTU2 (P=0.001, 0.041 and 0.002, respectively) in vitro (Fig. 3), further illustrating that invasiveness was dependent on strain. Our study appears to be the first to show that Ent. sakazakii invades capillary endothelial cells, suggesting that some strains from clusters 1 and 2 may be more likely to invade the BBB and cause CNS infection.
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). Strain NTU3 (16S rDNA cluster group 3) showed the least resistance to macrophage killing among Ent. sakazakii strains (Fig. 4). E. coli K1 (persists), E. coli K-12 (killed), and C. koseri strain 319 (replicates) were used as controls.
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). Bacteria that gain access to the bloodstream will be exposed to complement factors that cause cell lysis. Polymorphonuclear leukocytes are capable of releasing reactive oxygen species that damage cell membranes, proteins and essential macromolecules such as DNA. Ent. sakazakii strains were tested to determine resistance to acid, complement and reactive oxygen species. Cluster 3 and 4 strains and NTU1 were not resistant to serum complement (data not shown). All strains were resistant to acid at pH 4 except NTU1. Superoxide dismutases (SODs) are metalloenzymes which are found in all aerobic organisms and help protect bacteria from oxidative stress. All bacterial strains tested showed some level of SOD activity between 0.23 and 2.0 units ml–1 as assessed by the SOD assay (Fig. 5). Ent. sakazakii strains NTU1, NTU2, NTU3 and NTU84 demonstrated the highest SOD activity. Among Ent. sakazakii strains, NTU1 and NTU84 had significantly higher activity than NTU658 (P0.02) and NTU57 (P0.03). All the Ent. sakazakii strains exhibited SOD activity significantly lower than the positive control value (P0.05) that has a demonstrable affect on Y. entercolitica virulence (Roggenkamp et al., 1997); however, these studies demonstrate that SOD is functioning in Ent. sakazakii. While SOD is generally stationary-phase induced, and Fe-SOD is constitutively expressed, further sodA (Mn-SOD) activity may rely on specific induction such as acidic conditions that lead to oxidative stress (Kim et al., 2005). Deinococcus radiodurans is an example of a bacterium that can survive long term under harsh conditions, a property attributed to its strong SOD and DNA-repair activity (Tian et al., 2004). Ent. sakazakii is also known to survive long term in powdered infant formula (Caubilla-Barron & Forsythe, 2007). It is possible that SOD activity may contribute to this ability; this is currently under investigation.
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) and the finding of a partial gene sequence for sodA and sodB in the ongoing genome sequencing project (http://www.genome.wustl.edu) using the BLASTX program with homologous Salmonella Typhimurium LT2 sequences. The sodA and sodB genes are known to protect bacteria from oxidative stress (McCord & Fridovich, 1969) by catalysing the conversion of oxygen radicals to hydrogen peroxide (Farr & Kogoma, 1991). Sensitivity to oxidative stress may contribute to reduced virulence so we considered the capability of Ent. sakazakii to survive early contact with the macrophage oxidative burst (Beaman & Beaman, 1984) in addition to long-term persistence. While sodA was amplified from Ent. sakazakii cluster groups 1, 2 and 4 and C. koseri, it was not from cluster group 3 Ent. sakazakii strain NTU3 (data not shown). NTU3 was also the only strain found to be less numerous in macrophages at t24 compared to t0 (Fig. 4). However, the SOD assay results do not correspond to these data, in that NTU3 showed one of the highest SOD activity values (Fig. 5); this suggests that the poor ability of this strain both to survive early macrophage interactions and to replicate in macrophages may not be a result of a diminished SOD activity or lack of the sodA gene. The lack of amplification cannot rule out the presence of sodA in this strain, as it is a gene that exhibits a degree of variability. Considering the concern for rapid accurate identification of Ent. sakazakii it is of interest that the variability of sodA has recently been utilized in multiplex PCR reactions to identify enterococci at the genus and species level (Jackson et al., 2004). With respect to infection, inactivation of sodA in Y. enterocolitica resulted in a marked reduction in virulence of the organism in a mouse infection model (Roggenkamp et al., 1997). However, inactivation of sodA in Salmonella Typhimurium did not significantly attenuate infection in mice, which suggests that SodA is only required for initial resistance to macrophage oxidative burst. Studies utilizing deletion mutants are ongoing to clearly define the importance of sodA during Ent. sakazakii infection.
In E. coli K1 the ompA gene has been linked with serum resistance and CNS invasion in the neonatal rat model (Weiser & Gotschlich, 1991). The ompA gene was not amplified from cluster 4 strain NTU84 (data not shown). Our studies also show that NTU84 is serum sensitive. Since OmpA is reported to contribute to serum resistance we sought to determine if the presence of ompA among cluster 4 strains was associated with serum sensitivity. However, all other cluster 4 strains had the ompA gene and these strains were serum sensitive (data not shown), suggesting that OmpA may not play an essential role in Ent. sakazakii serum resistance. OmpA has been associated with increased HBMEC invasion in vitro (Shin et al., 2005). NTU84 had relatively low invasive abilities in our endothelial cell line, suggesting that OmpA may influence capillary endothelial cell invasion. Alternatively, analogous mechanisms for resistance and invasion may not be present in NTU84. The development and specificity of the ompA PCR as a potential tool for the rapid detection of Ent. sakazakii in infant formula is impressive (Mohan Nair & Venkitanarayanan, 2006). However, the finding of an Ent. sakazakii strain (NTU84) that is not amplified with this method suggests that ompA PCR would not detect Ent. sakazakii in every case.
Cytokine secretion from U937 macrophages containing Ent. sakazakii
Since macrophages are thought to be early regulators of the innate immune response that further dictates the adaptive immune response, cytokine secretion from macrophages was assessed via ELISA following Ent. sakazakii inoculation. TNF levels were not significantly different from 6 to 24 h and strain NTU84 was most robust, secreting 500 pg TNF ml–1 after 24 h (Fig. 6a). IL-6 levels more than doubled and again strain NTU84 had the most robust response, secreting nearly 1200 pg IL-6 ml–1, indicative of a strong inflammatory response elicited from macrophages in response to NTU84 infection (Fig. 6b). Both patterns of cytokine expression match those expected: TNF expression occurs rapidly and levels out while IL-6 gradually increases over time. IL-10 secretion was induced (Fig. 6c); however, IL-12 was not recovered in significant amounts (data not shown). Cytokine levels in serum collected from neonatal rats 24 h following i.p. inoculation with strain 2 were also tested using ELISA. The level of IL-10 elicited by strain 2 was 562±133 pg ml–1. The level of IL-6 was 2199±497 pg ml–1. The type 2 immune response is induced when the IL-10/IL-12 ratio is high, because anti-inflammatory IL-10 can suppress pro-inflammatory IL-12. This response is insufficient at clearing intracellular infections. An early bias towards a type 2 immune response may contribute to an inability to successfully eliminate this intracellular pathogen.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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Received 26 April 2007;
revised 12 June 2007;
accepted 29 June 2007.
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