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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Feb 7;104(7):2396–2401. doi: 10.1073/pnas.0610833104

O-Specific polysaccharide conjugate vaccine-induced IgG antibodies prevent invasion of Shigella into Caco-2 cells and may be curative

Yehuda Chowers *,†,‡, Joachim Kirschner §, Nathan Keller †,, Iris Barshack †,‖, Simon Bar-Meir *,†, Shai Ashkenazi †,**, Rachel Schneerson ††, John Robbins ††,‡‡, Justen H Passwell †,§§,¶¶
PMCID: PMC1892925  PMID: 17287349

Abstract

The O-specific polysaccharide (O-SP) domain of Shigella LPS is both an essential virulence factor and a protective antigen for this genus. A critical level of serum IgG anti-O-SP was shown to confer immunity to shigellosis, likely by complement-mediated bacteriolysis of the inoculum. Conjugate Shigella O-SP vaccines were shown to be safe and immunogenic in children, and, in a preliminary study, Shigella sonnei vaccine was protective in young adults. Characteristic of shigellosis is bacterial invasion of intestinal cells. Incubation of shigellae with postimmunization but not preimmunization sera of children vaccinated with S. sonnei or Shigella flexneri 2a O-SP conjugate vaccines inhibited in a type-specific and dose-dependent manner in vitro invasion of intestinal epithelial cells (Caco-2) and the infection-associated increases in IL-1β and IL-8 mRNA and extracellular cytokine levels. Pretreatment of these sera or of Caco-2 cells with O-SP abrogated these effects also in a type-specific and dose-dependent manner. Confocal microscopy demonstrated antibody-specific inhibition of bacterial adhesion to HeLa cells. These protective effects were duplicated by IgG purified from these sera. These results suggest a dual role for IgG anti-O-SP. In addition to lysis of the inoculum in immune individuals, the newly synthesized IgG anti-O-SP in patients may terminate an established infection by inhibiting shigellae released from epithelial cells from invading new ones. A critical level of IgG anti-O-SP could, therefore, have a protective as well as a curative role in shigellosis.

Keywords: vaccination

Shigellae are a major cause of endemic and epidemic dysentery, especially in developing countries (1). Shigellae enter the mucosa via M cells (2), infect resident macrophages causing their apoptosis, then enter the epithelial cells via their basolateral surfaces (3, 4). Infection of macrophages and epithelial cells results in secretion of proinflammatory cytokines (5–7), which are responsible for tissue damage and clinical symptoms.

O-specific polysaccharide (O-SP), the outermost domain of the LPS of shigellae is both an essential virulence factor and a protective antigen based on the following evidence: (i) Shigellae from patients' stool invariably express a complete LPS, whereas LPS-deficient strains are avirulent (8); (ii) the closely related Escherichia coli that express Shigella LPS assume the virulence of this species (9); (iii) convalescence from disease confers LPS-specific immunity, albeit incomplete and of limited duration (1, 10); (iv) preexisting IgG LPS antibodies protected soldiers from shigellosis (11); and (v) immunization of soldiers with a Shigella sonnei conjugate conferred protection against this pathogen related to the level of vaccine-induced IgG antibodies (12). Similar to capsulated pathogens, such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, there is an inverse relation between the level of “natural” anti-LPS and the age incidence of shigellosis. Shigellosis occurs rarely below the age of 6 months, peaks at the ages between 1 and 6 years, and declines in older children and adults (13, 14).

Based on these observations, conjugate vaccines aimed to prevent shigellosis in young children are being evaluated. Safety and immunogenicity (phase 2) studies have been successfully completed in 4- to 7-year-old (15) and 1- to 4-year-old children (16). In the present study, we used immune sera from these children to examine their effect on the invasion of epithelial cells by shigellae and the ensuing inflammatory response by using an in vitro model of bacterial invasion into Caco-2 cells.

Results

Immune Sera Prevent Invasion of Shigella into Caco-2 Cells.

Immune sera from four children immunized with the S. sonnei conjugate and from four children immunized with the Shigella flexneri 2a conjugate significantly inhibited invasion of intestinal epithelial cells (Caco-2) by the homologous bacteria (see Table 1 for antibody levels). No effect was noted either with matched preimmune sera (containing little anti-O-SP) or with heterologous immune sera, indicating that the protective effect was due to type-specific antibodies (Fig. 1). Incubation of S. flexneri 2a with a serially diluted immune serum showed an inverse relation between the amounts of serum added and bacterial invasion (Fig. 2). Similar results were obtained with S. sonnei (data not shown).

Table 1.

Antibody levels of sera used in this study

Volunteers and vaccines Anti-S. sonnei IgG Anti-S. flexneri2a IgG
1
    S. sonnei A 1.1 1.2
    S. sonnei C 84.6 1.2
2
    S. sonnei A 1.0 1.3
    S. sonnei C 103.2 1.2
3
    S. sonnei A <0.5 2.3
    S. sonnei C 51.2 2.4
4
    S. sonnei A 0.6 2.5
    S. sonnei C 96.9 2.8
5
    S. sonnei A 0.5 2.2
    S. sonnei C 121.8 2.6
6
    S. flexneri 2a A <0.5 1.1
    S. flexneri 2a C 0.8 29.3
7
    S. flexneri 2a A <0.5 1.3
    S. flexneri 2a C <0.5 66.8
8
    S. flexneri 2a A <0.5 1.2
    S. flexneri 2a C <0.5 71.5
9
    S. flexneri 2a A <0.5 1.5
    S. flexneri 2a C <0.5 51.6
10
    S. flexneri 2a A <0.5 1.8
    S. flexneri 2a C <0.5 423.5
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Volunteers 1–5 were immunized with S. sonnei vaccine, and volunteers 6–10 were immunized with S. flexneri 2a vaccine. Results are given for measurements taken before (A) and 4 weeks after (C) the second immunization with the respective O-SP-conjugate vaccine. Antibody levels to the LPS of S. sonnei and S. flexneri 2a were measured by ELISA referred to a standard serum and are expressed in ELISA units.

Fig. 1.

Fig. 1.

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Effect of S. flexneri 2a and S. sonnei immune sera on bacterial invasion into Caco-2 cells. Caco-2 cells were grown as confluent monolayers. Bacteria were treated with either homologous or heterologous, preimmune or immune sera as described in Materials and Methods. Preimmune sera, cross-hatched bars; immune sera, black bars. Volunteers 1–4 were immunized with the S. sonnei conjugate. (A) Treatment and inoculation of S. sonnei. (B) Treatment and inoculation of S. flexneri 2a. Volunteers 6–9 were immunized with the S. flexneri 2a conjugate. (C) Treatment and inoculation of S. flexneri 2a. (D) Treatment and inoculation of S. sonnei. (A and C) The results are presented as percentages of bacterial invasion in controls without serum for all pairs. (Differences between OD measurements: preimmune vs. immune, P < 0.001.)

Fig. 2.

Fig. 2.

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Dose–response curve of the protective effect of immune serum. The immune serum from volunteer 6 was serially diluted to a final volume of 100 μl. S. flexneri 2a bacteria were treated with the serum dilutions and added to cultured Caco-2 cells. The volume of added serum is indicated. After incubation, the cells were lysed, and the lysates were inoculated into liquid medium. Bacterial growth was assessed by OD600. Averages of three experiments, each performed in duplicate, are presented as percent of control (untreated bacteria). (Differences between OD measurements: 2.5 μl vs. untreated, P < 0.001.)

Adsorption of Immune Sera with O-SP Abrogates Their Effect on Bacterial Invasion into Caco-2 Cells (Fig. 3).

Fig. 3.

Fig. 3.

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Effect of the pretreatment of immune sera with O-SP on bacterial invasion into Caco-2 cells. Immune sera (black bars) from volunteer 2 immunized with S. sonnei conjugates (A) and from volunteer 6 immunized with S. flexneri 2a conjugates (B), preincubated with the immunizing type O-SP, and unadsorbed preimmune sera (white bars) were infected with their respective bacteria and inoculated into Caco-2 cell cultures (see Materials and Methods). The results are compared with those from untreated bacteria (gray bars). Immune sera preincubated with the heterologous O-SP (cross-hatched bars) served as controls. After culture, cell lysates were cultivated in BHI, and bacterial growth was assessed by OD600. The mean and SD of three experiments, each performed in duplicate, are presented [no serum vs. immune serum adsorbed with the homologous O-SP, P = 0.29 (not significant) for both shigellae].

To verify the specificity of protection afforded to Caco-2 cells by immune sera, these sera were adsorbed with homologous or heterologous O-SP. Unadsorbed preimmune sera had little effect on bacterial invasion of Caco-2 cells; growth was similar to that of bacteria only. The immune serum-mediated inhibition of bacterial invasion of Caco-2 cells was reduced proportionally to the amount of homologous O-SP used for serum adsorption. In contrast, preincubation with the heterologous O-SP had no effect; the serum still exerted full inhibition of bacterial invasion.

IgG from Immune Sera Mimics the Effect of the Sera.

Depletion of IgG from the immune sera, abolished their effect on invasion of Caco-2 cells by Shigella (data not shown). IgG from the immune sera inhibited invasion of Caco-2 cells in a dose-dependent manner (Fig. 4A). Fig. 4 B and C shows a type-specific and dose-dependent abrogation of the protective effect when the IgG was preincubated with increasing amounts of the homologous O-SP. O-SP from the heterologous strain had no effect.

Fig. 4.

Fig. 4.

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Effect of isolated IgG on bacterial invasion into Caco-2 cells. Shigellae were treated with IgG from S. sonnei (volunteer 5) or S. flexneri 2a (volunteer 10) vaccinees. (A) Dose–response to homologous IgG added to shigellae cultures. (B) Effect of anti-S. sonnei IgG adsorbed with increasing concentrations of S. sonnei or S. flexneri 2a O-SP on S. sonnei invasion of Caco-2 cells. (C) Effect of anti-S. flexneri 2a IgG adsorbed with increasing concentrations of S. flexneri 2a or S. sonnei O-SP on S. flexneri 2a invasion of Caco-2 cells. Results are relative to those with untreated bacteria and presented as the mean and SD of three experiments, each performed in triplicate [preimmune or heterologous IgG vs. homologous immune IgG (both serotypes), P < 0.001].

Treatment of Caco-2 Cells with O-SP Prevents Their Invasion by Shigella.

Preincubation of Caco-2 cells with S. sonnei O-SP blocked their invasion by S. sonnei in a dose-dependent and type-specific manner (Fig. 5A). In contrast, S. flexneri 2a O-SP had no such effect on invasion by S. sonnei and vice versa (Fig. 5B). These observations suggest that invasion of Caco-2 cells by Shigella requires binding of their O-SP to the epithelial cells and that blocking this binding is a protective mechanism.

Fig. 5.

Fig. 5.

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Effect of pretreatment of Caco-2 cells with O-SP on bacterial invasion. Caco-2 cells were pretreated with increasing concentrations of S. sonnei or S. flexneri 2a O-SP before the addition of bacteria. (A) Inoculation with S. sonnei. (B) Inoculation with S. flexneri 2a. Black bars, cells treated by the immunizing O-SP; cross-hatched bars, treatment with the heterologous O-SP; gray bars, untreated cells. After treatment, the cells were inoculated with bacteria, and intracellular bacteria were quantified by cfu counts (see Materials and Methods). Results are the mean and SD of two experiments performed in duplicate (S. sonnei vs. S. sonnei plus 50 μg of S. sonnei O-SP and S. flexneri 2a vs. S. flexneri 2a plus 50 μg of S. flexneri 2a O-SP, P < 0.001).

Immune Sera Prevent Secretion of IL-8 and IL-1β (Fig. 6).

Fig. 6.

Fig. 6.

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Expression of IL-8 and IL-1β mRNA by Caco-2 cells after invasion by bacteria treated with preimmune and immune sera. Bacteria were pretreated with preimmune or immune sera to S. sonnei (volunteer 2) and then added to Caco-2 cultures for 1 h; the medium was changed, and the cultures were continued for an additional 1 h. The cells were then washed, and RNA was extracted and analyzed by an RNA protection assay. Lanes: A, medium only; B, untreated S. sonnei; C, S. sonnei treated with preimmune serum; D, S. sonnei treated with immune serum.

Invasion of shigellae into intestinal epithelial cells induces secretion of IL-8 and other proinflammatory cytokines (7, 17). As shown in Table 2, invasion of the bacteria induced secretion of both IL-8 and IL-1β from Caco-2 cells. This effect was inhibited by the immune sera, whereas preimmune sera were not inhibitory.

Table 2.

Secretion of IL-8 and IL-1β by Caco-2 cells after invasion by bacteria treated with preimmune and immune sera

Infecting bacteria Treatment IL-8, ng/ml IL-1β, ng/ml
None Medium 13.26 ± 3 0
S. sonnei Medium 116.9 ± 8.8 1.51 ± 0.01
S. sonnei S. sonnei preimmune 114.3 ± 14.9 1.38 ± 0.2
S. sonnei S. sonnei immune 18.4 ± 0.6 0.01
S. sonnei S. flexneri 2a immune 107.5 ± 3.6 1.58 ± 0.06
S. flexneri 2a Medium 119.9 ± 17.3 1.54 ± 0.02
S. flexneri 2a S. flexneri 2a preimmune 118.53 ± 7.46 1.56 ± 0.03
S. flexneri 2a S. flexneri 2a immune 17.62 ± 1.1 0
S. flexneri 2a S. sonnei immune 128.5 ± 1.3 1.56 ± 0.02
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Bacteria were pretreated with preimmune or immune sera to S. sonnei (volunteer 2) or S. flexneri 2a (volunteer 6) and then added to Caca-2 cells as described. The supernatants were assayed by ELISA for IL-8 and IL-1β concentrations. The results are expressed as the means and SEs of three experiments performed in triplicate (IL-8 and IL-1β preimmune vs. immune, P < 0.001).

Expression of IL-8 and IL-1β mRNA by Caco-2 cells was increased after invasion of the shigellae organisms. Pretreatment of the bacteria with specific immune sera resulted in reduced expression of both mRNAs, confirming the protective effect of the immune sera against bacterial invasion.

Confocal Microscopy Confirms Inhibition of Invasion by Immune Serum (Fig. 7).

Fig. 7.

Fig. 7.

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The effect of immune sera on bacterial adhesion to HeLa cells. HeLa cells were infected with S. flexneri 2a and S. sonnei in the presence or absence of their respective immune serum. The cells were then fixed and stained for actin with TRITC-phalloidin and for DNA with DAPI. The first row shows untreated cells, the second row shows cells infected with S. flexneri 2a, and the third row shows cells infected with S. flexneri 2a in the presence of an immune S. flexneri 2a serum. Row four shows cells infected with S. sonnei, and row five shows cells infected with S. sonnei in the presence of S. sonnei immune serum. The first column depicts actin staining, the second column depicts the DAPI staining, and the third column is a superposition of the two. (Scale bar, 20 μm.)

To test whether bacterial adherence to the epithelial cells was inhibited by immune serum, HeLa cell cultures inoculated with bacteria in the presence of preimmune and immune sera were examined by confocal microscopy. Treatment with immune sera conferred type-specific inhibition of bacterial adhesion to the cells. No effect was observed with preimmune or heterologous immune sera (data not shown).

Discussion

We present evidence that IgG anti-O-SP may terminate an established infection as well as prevent shigellosis. Conjugate vaccine-induced IgG anti-O-SP protected Caco-2 epithelial cells from invasion and prevented the secondary provoked secretion of IL-8 and IL-1β by shigellae in a type-specific and dose-dependent manner. Sera depleted only of their IgG lost this protective effect, and isolated IgG reproduced the activity of whole serum.

An intact LPS is necessary for the virulence of shigellae; strains deficient in O-SP are not virulent, and serum IgG antibodies to the O-SP confer immunity. The symptoms of shigellosis, including fever and dysentery (blood and mucus in the stool and cramps), are mediated by the lipid A region of LPS but it is the O-SP that both mediates the invasion of epithelial cells by shigellae and is a protective antigen. The prevention of invasion of Shigella into Caco-2 cells by type-specific (i.e., O-SP-specific) sera or IgG and the abrogation of this effect by adsorption of the sera with the O-SP or by incubation of Caco-2 cells with purified O-SP, thereby blocking O-SP on the bacteria from binding to the epithelial cells, provide mechanisms of pathogenicity and protection. We hypothesized that a critical level of serum IgG anti-LPS exudes onto the epithelium of the intestine (13, 18). Complement proteins are also present on the epithelial surface (19). This combination of IgG anti-O-SP and complement could result in bacteriolysis of the inoculum of shigellae. Inhibition of invasion of shigellae into epithelial cells, where they are shielded from antibody and complement, renders them susceptible to lysis by IgG anti-O-SP and complement. This combination breaks the vicious cycle of cell invasion and bacterial proliferation that allows for the mucosal spread of infection, which also is the likely mechanism by which an established infection is cured, given that synthesis of IgG anti-LPS follows acute infection. Serum depleted only of its IgG lost its protective effect, and isolated IgG reproduced the effect of the whole serum, confirming its specific importance. Our finding that treatment of Caco-2 cells with the O-SP inhibited invasion by shigellae was unexpected. This inhibition was specific and quantitative and suggests that there is a binding site for O-SP on epithelial cells, explaining the virulence properties of shigellae. This interaction should be studied further, especially with an innate immune system such as the Toll-like proteins.

Cytokine secretion that activates NF-κB signal transduction, plays an important role in mucosal damage after Shigella infection (7, 20). A major consequence of this activation is secretion of IL-8, which recruits polymorphonuclear leukocytes, with resultant inflammation (5). IL-1β was also shown to play an important role in the pathogenesis of Shigella infection (21, 22). Our observations indicate that IgG anti-O-SP abrogates transcription of IL-1β and IL-8. Inhibition of invasion of Caco-2 cells by shigellae and the secondary biologic effects thereof may prevent tissue damage and further spread of the Shigella within mucosal cells. As a result, the extracellular shigellae will now be susceptible to antibody-mediated complement-dependent bacteriolysis. The action of IgG anti-O-SP may therefore be considered both as a preventive and a curative mechanism.

Materials and Methods

Materials.

Brain–heart infusion (BHI), Salmonella–Shigella agar, and McConkey agar plates were from Hy Laboratories (Rehovot, Israel). Caco-2 cells and HeLa cells were from the American Type Tissue Collection (Rockville MD). DMEM, calcium-free DMEM, F-12HAM, EGTA, Moviol, TRIT-C-phalloidin/DAPI, paraformaldehyde, trypsin, glutamine, penicillin, streptomycin, gentamycin, and p-nitrophenylphosphate were from Sigma (St. Louis, MO). PBS and FCS was from GIBCO/BRL (Auckland, New Zealand). The EZ PCR mycoplasma kit was from Biological Industries (Beit Haemek, Israel). The BBL Crystal TM Enteric/Nonfermentor ID system was from Becton Dickinson (Cockeysville, MD), and Shigella type-specific antisera were from the National Center for Enteric Pathogens (Jerusalem, Israel). Tissue culture plates were from Sarstedt (Nümbrecht, Denmark). The Mab Trap kit was from Amersham (Uppsala, Sweden). The ELISA kit for serum Igs was from Bethyl Laboratories (Montgomery, TX). IL-8 ELISA was from R&D Systems (Minneapolis, MN), and IL-1β was from Genzyme (Cambridge, MA). The RNA-extraction Tri reagent kit was from MRC (Cincinnati, OH). The Tri reagent kit and the RiboQuant multiprobe RNA protection assay were from Pharminigen (San Diego, CA).

Sera.

Sera were from 1- to 4-year-old children immunized with investigational S. sonnei and S. flexneri 2a conjugate vaccines (16). Each child received two injections of one of the two vaccines, with 6 weeks between vaccinations. IgG anti-LPS was measured by ELISA (23) in sera taken before weeks 1 and 4 after the second injection. Antibody levels of the sera used in this report are shown in Table 1. IgG was isolated from the same sera with an affinity chromatography Mab Trap kit according to the manufacturer's instructions. The purity of the preparations was verified by measuring the serum Ig fractions with an Immage instrument (Beckman immunochemistry system) and a single immunodiffusion assay and by ELISA.

Bacteria.

Strains of S. sonnei and S. flexneri 2a from patients with dysentery were obtained from the Department of Clinical Microbiology, Sheba Medical Center and identified with the BBL Crystal TM Enteric/Nonfermentor ID system and by agglutination with type-specific antisera. Bacteria were frozen at −70°C until used.

Bacteria were grown on Salmonella–Shigella agar plates for 1 day, then inoculated into BHI media and grown overnight. Before inoculation into the Caco-2 cell cultures, the bacteria were transferred into fresh BHI media and grown to an OD600 of 0.5.

LPS and O-SP.

LPS and O-SP were isolated as described in ref. 23.

Cell Lines and Culture.

Caco-2 cells were maintained at 37°C and 5% CO2 in DMEM nutrient mixture with F-12 Ham supplemented with 10% FCS, 1% glutamine, and 1% penicillin and streptomycin. HeLa cells were maintained in DMEM nutrient mixture with F-12 Ham at 37°C and 5% CO2. Test of cultures for Mycoplasma infection using EZ PCR mycoplasma test kits was negative.

Infection of Cells.

Caco-2 cells were infected with shigellae as previously described (24) with slight modifications as follows. Cells were transferred into 24-well plates and grown to semiconfluency. Four hours before inoculation with bacteria, the medium was changed to calcium-free DMEM, after which the cells were treated for 1 h with 100 μM EGTA. Thereafter, Shigella was added to the culture in 1-ml increments at a concentration of 2.6 × 106 cfu/ml and incubated at 37°C for 1 h. Subsequently, the cells were washed four times with fresh medium containing 50 μg/ml gentamycin, and the culture continued for an additional 2 h. Thereafter, the cells were washed three times with PBS and lysed with deionized water. The lysate was inoculated into BHI medium for 20 min at 37°C with shaking, and bacterial growth was assessed at OD600. In other experiments, bacteria were diluted serially and plated onto McConkey agar plates, and the colonies were counted. HeLa cells were inoculated with bacteria as described for the Caco-2 cells. After a 1-h incubation, the cells were washed with PBS, then permeabilized for 2 min with 3% paraformaldehyde/0.5% Triton X-100 and fixed for 20 min with 3% paraformaldehyde. The coverslips were then incubated for 40 min with TRIT-C-phalloidin/DAPI, washed for 3 × 10 min with PBS, and embedded in Elvanol. Elvanol was prepared by dissolving 25 g of Moviol in 100 ml of 0.15 M PBS and stirring for 16 h at room temperature. Then 50 ml of glycerol was added, and stirring continued at room temperature for another 16 h. The mixture was then centrifuged for 15 min at 12,000 rpm (Centrifuge 5417R; Eppendorf, Hamburg, Germany), and the supernatant was used for embedding.

Treatment of Bacteria with Sera.

Serum (10 μl) or IgG (10 μl) was added to 90 μl of overnight bacterial cultures containing 2.6 × 107 cfu/ml for 1 h at 37°C.

Treatment of Sera with O-SP.

Immune sera and isolated IgG were first incubated with various amounts of S. Sonnei or S. flexneri 2a O-SP for 1 h at 37°C, then added to bacteria at a final concentration of 2.6 × 106 cfu/ml for an additional 1 h, after which the bacteria were incubated with Caco-2 cells as described.

Treatment of Caco-2 Cells with O-SP.

After changing the medium to calcium-free DMEM (as above) and before the addition of bacteria, Caco-2 cells were treated with either S. sonnei or S. flexneri 2a O-SP for 1 h at 37°C. Subsequently, 0.5 ml of bacterial suspension containing 108 cfu/ml was added, and the experiment continued as described above.

Cytokine ELISA, RNA Extraction, and RNA Protection Assay.

Bacteria were added to Caco-2 cells as described under Infection of Cells. The cells were washed four times with medium containing gentamycin, and the culture continued for a total of 24 h. The supernatants were collected and assayed for IL-8 and IL-1β concentrations by ELISA (25). Briefly, 96-well plates were coated with polyclonal goat anti-human IL-8 as capturing antibodies. After incubation with the cell culture supernatants and washing, polyclonal rabbit anti-human antibodies were added. Alkaline phosphatase-conjugated mouse anti-rabbit IgG was used as a secondary antibody. The assay was developed by using p-nitrophenylphosphate substrate.

For mRNA expression, Caco-2 cells were grown to semiconfluency in 10-cm tissue culture plates. Bacteria, untreated or treated with serum, were added to the culture for 1 h, after which the cells were washed four times with medium containing gentamycin. The cell culture was then continued for an additional 1 h and treated with trypsin for 2 min. Thereafter, the cells were washed in DMEM/F-12 medium, spun for 5 min at 1,600 rpm (Eppendorf), and the RNA was extracted from the pellet. mRNA levels were measured by the RiboQuant multiprobe RNA protection assay by following the manufacturer's instructions.

Statistics.

Data are presented as means ± SE. All significance tests were performed with a two-tailed Student t test. Logarithmic transformation was used where appropriate. P values of <0.05 were considered significant.

Acknowledgments

We thank A. Ginzberg for diligent and careful assistance with graphics. This work was supported by National Institute of Child Health and Human Development Contract N01-HD-5-3226, The Intramural Research Program of the National Institute of Child Health and Human Development, and the Centre for the Study of Emerging Diseases (Jerusalem, Israel).

Abbreviations

O-SP

O-specific polysaccharide

HBI

heart–brain infusion.

Footnotes

The authors declare no conflict of interest.

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