Pneumococcal agglutination by anti-capsule antibodies can be quantified using flow cytometry.

<p>Agglutination with two pneumococcal strains (TIGR4 and EF3030) incubated with different concentrations of serotype-specific rabbit antiserum (serotype 4 or serotype 19F) and rabbit antiserum against a heterologous serotype (serotype 14) (performed in duplicate). Individual histograms of a representative measurement are shown for the forward scatter of TIGR4 and for EF3030 following incubation with type-specific antiserum <b>(A),</b> overlays of the different concentrations of type-specific antiserum are shown <b>(B)</b> and all data (type-specific and heterologous antiserum) is summarized in a single graph <b>(C)</b>. The dashed line represents the agglutination cutoff value.</p

Agglutination of <i>S</i>. <i>pneumoniae</i> by anti-capsule antibodies can be detected by flow cytometry.

<p><i>S</i>. <i>pneumoniae</i> TIGR4 were incubated with a serotype specific polyclonal rabbit antiserum (serotype 4; α-ST4; right panel) and with rabbit antiserum against a heterologous serotype (serotype 14; α-ST14; left panel). Agglutination was assessed by flow cytometry and is represented as dot plots of the FSC-A vs the SSC-A <b>(A)</b> and histograms of the FSC-A signal <b>(B)</b> and by phase contrast microscopy <b>(C)</b>.</p

Exogenous hFH fails to attenuate disease scores, inflammatory cytokine production, and vascular leakage in the liver.

<p>Mice infected with 1x10⁷ CFU of <i>S</i>. <i>pneumoniae</i> (TIGR4) and sham infected control mice were all treated with antibiotics at t = 17 h and indicated groups received an injection with hFH or PBS as control (n = 10). The disease score was monitored at t = 17, t = 21 and t = 26 hours after inoculation (A). The black bar represents uninfected mice, gray bar infected control mice and white bar hFH treated mice. Data points represent the median value with interquartile range. At t = 26 h, serum pro-inflammatory cytokines IL-6 and MIP-2 were measured by ELISA (B, C). Liver vascular leakage was measured by Evans Blue-albumin extravasation to quantify vascular permeability (D). Raw fluorescence intensities (RFI) were recorded and multiplied by the wet organ weight to estimate the concentration of Evans Blue in the organ. Each point depicted in graphs B,C and D indicates one mouse. One infected mice of the PBS treated group reached the humane endpoint at t = 22 h and was excluded from the graphs. Furthermore one (IL-6 Fig 1B) respectively two data points (MIP-2 Fig 1C) are missing, as insufficient serum was available. In addition, one data point is missing in the vascular leakage graph, because of a technical failure during injection of EB in one mouse. Cytokine values were analyzed after logarithmic transformation; the horizontal line represents the median. Dash line indicates lower limit of detection. Comparison between groups were performed by using the non-parametric Mann-Whitney test with Bonferroni correction * p < 0.05 was considered significant. ** p< 0.01, *** p<0.001, ns = not significant.</p