Extracellular levels of granzyme A and B on admission and during discharge in patients.

<p>Typhoid fever patients (n = 15) who were discharged from hospital had lower levels of granzymes at follow-up when patients were clinically improved (granzyme A; A), although this did only reach statistical significance for granzyme B (B). Medians are shown. Significance determined via Mann-Whitney <i>U</i> tests. *P<0.05. Gzm: granzyme.</p

Proportion and cell counts of lymphocyte populations and granzymes in patients with typhoid fever and controls.

<p>Proportion and cell counts of lymphocyte populations and granzymes in patients with typhoid fever and controls.</p

Extracellular levels of granzyme A and B in patients with typhoid fever and healthy controls, and correlation with IFN- γ levels.

<p>Plasma levels of granzyme A (A) and B (B) measured in healthy controls (n = 38) compared to admission samples of hospitalized typhoid fever patients (n = 28). Medians are shown. Significance determined via Mann-Whitney <i>U</i> tests. ***P<0.001. Granzyme A is correlated to granzyme B (C). Levels of granzyme A (D) and granzyme B (E) are correlated to interferon (IFN)-γ in patients. Correlation coefficient reported is for Spearman's Rho. Gzm: granzyme.</p

Expression of granzymes A, B and K by each lymphocyte subtype in patients with typhoid fever and controls.

<p>Percentage of lymphocyte population expressing each granzyme (A–D) and MFI of the intracellular expression of each granzyme in the lymphocyte populations (E–H). n = 36 controls and 8 patients. MFI values are expressed with Log10 scale. Significance determined via Mann-Whitney <i>U</i> tests. Medians are shown. Analysis was done by flow cytometry. MFI: median fluorescence intensity; NK cells: CD3⁻CD56⁺. *P<0.05, **P<0.01, ***P<0.001.</p

Demographic, clinical and laboratory features of typhoid fever patients compared to controls.

<p>Demographic, clinical and laboratory features of typhoid fever patients compared to controls.</p

Immunostaining for S100A9 in the liver and spleen of mice during <i>S</i>. Typhimurium infection.

<p>Positive immunostaining for S100A9 in liver parenchyma (A) and the red pulp of the spleen (C) tissue was observed in uninfected control animals. Five days after infection with <i>S</i>. Typhimurium (10⁶) there was a marked increase of S100A9 in both liver (B) and spleen (D) corresponding with inflammatory cell influx. Magnification 10 × and 40 ×.</p

Patient characteristics.

<p>Baseline characteristics of patients with acute typhoid fever (admitted to the hospital, Bangladesh study) or during convalescence (treated in an outpatient setting, Indonesian study). Days of fever prior to admission or when sample was taken, median. Specific complications were: pneumonia (n = 3); encephalopathy (n = 2); gastrointestinal bleeding (n = 1); severe anemia requiring blood transfusion (n = 1); acute hepatitis (n = 1); hepatic abscess (n = 1); septic arthritis (n = 1); and renal failure, cardiopulmonary arrest and death (n = 1). N/A: not applicable.</p><p>Patient characteristics.</p

S100A8/A9 complexes are abundantly present in plasma and feces from patients with typhoid fever and correlate with duration of fever.

<p>Increased levels of S100A8/A9 (ng/ml) complexes are measured in plasma of patients with confirmed typhoid fever caused by <i>S</i>. Typhi admitted to the hospital (A; n = 28 for admission) and compared to healthy controls (n = 38). However S100A8/A9 levels were not yet normalized at time of discharge (n = 15). In a community based control study during convalescence (3 weeks post infection) S100A8/A9 remains elevated (B; n = 60) compared to healthy controls (n = 60). Increased levels of S100A8/A9 (μg/g) measured in feces from hospitalized patients (C; n = 14) compared to healthy controls (n = 36). No differences were seen in levels of S100A8/A9 between hospitalized patients with complicated or uncomplicated typhoid fever as detailed in method section (D). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation. Correlation curve log alanine aminotransferase (ALT) versus log plasma S100A8/A9 (E). Correlation curve days of fever prior to admission versus log plasma S100A8/A9 (F). For linear regression analysis, P value and Spearman rho are reported.</p

S100A8/A9 inhibits bacterial growth of <i>S</i>. Typhimurium and <i>S</i>. Typhi in vitro.

<p>Growth of <i>S</i>. Typhimurium (14028; A) and <i>S</i>. Typhi (Vivotif, Crucell; B) was assessed for a maximum of 24 hours in the presence of recombinant S100A8/A9 (50 μg/ml; grey bars) or control (white bars). Bacterial growth was dose dependently inhibited by S100A8/A9. Growth arrested, CFSE-labelled heat-inactivated <i>S</i>. Typhimurium bacteria were incubated with peripheral blood neutrophils (C) from wildtype (WT; white) and <i>S100A9</i>^<i>-/-</i> mice (grey) (n = 4 per mouse strain) for 0, 15 and 60 minutes respectively after which phagocytosis was quantified (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003663#sec005" target="_blank">Methods</a>). Data are expressed, as mean ± SD. **** P<0.0001, determined via non-parametric <i>t</i> tests comparing different dosages per time point.</p

No effect of S100A8/A9 deficiency on organ injury during <i>S</i>. Typhimurium infection.

<p>Representative slides of liver (A, B) and spleen (D, E) hematoxylin and-eosin (HE) staining of wildtype (WT) and <i>S100A9</i>^<i>-/-</i> mice, 5 days post infection. Liver and spleen from both WT and <i>S100A9</i>^<i>-/-</i> mice displayed advanced inflammation with necrosis (#) and thrombosis (*). Magnification 10 ×. Total pathology score was determined at indicated time points in WT (white bars) and <i>S100A9</i>^<i>-/-</i> (grey bars) mice according to the scoring system described in the methods section (C, F). Aspartate aminotransferase (AST; G), alanine aminotransferase (ALT; H), and lactate dehydrogenase (LDH; I) were measured in plasma. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (5–8 mice per group at each time point).</p