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

Short-term repeated HRV-16 exposure results in an attenuated immune response <i>in vivo</i> in humans - Fig 5

<p>Plasma IP-10 levels following placebo inoculation and HRV challenge (panel A) and following two HRV challenges separated by one week time (panel B). Panel C shows the peak levels in the first four days post-challenge in the group that received placebo, followed by a HRV challenge (bar 1 and 2) and in the group that were challenged with HRV twice (bar 3 and 4). Data are represented as geometric mean and 95% CI. Lower detection limit was 156 pg/mL.</p

Short-term repeated HRV-16 exposure results in an attenuated immune response <i>in vivo</i> in humans

<div><p>Introduction</p><p>Naturally, development of adaptive immunity following HRV infection affects the immune response. However, it is currently unclear whether or not HRV re-exposure within a short time frame leads to an altered innate immune response. The “experimental cold model” is used to investigate the pathogenesis of HRV infection and allows us to investigate the effects of repeated exposure on both local and systemic innate immunity.</p><p>Methods</p><p>40 healthy male and female (1:1) subjects were nasally inoculated with HRV-16 or placebo. One week later, all subjects received HRV-16. Baseline seronegative subjects (n = 18) were included for further analysis.</p><p>Results</p><p>Infection rate was 82%. Primary HRV infection induced a marked increase in viral load and IP-10 levels in nasal wash, while a similar trend was observed for IL-6 and IL-10. Apart from an increase in IP-10 plasma levels, HRV infection did not induce systemic immune effects nor lower respiratory tract inflammation. With similar viral load present during the second HRV challenge, IP-10 and IL-6 in nasal wash showed no increase, but gradually declined, with a similar trend for IL-10.</p><p>Conclusion</p><p>Upon a second HRV challenge one week after the first, a less pronounced response for several innate immune parameters is observed. This could be the result of immunological tolerance and possibly increases vulnerability towards secondary infections.</p></div

RSV neutralization does not correlate with severity of disease.

<p>50% plaque reduction neutralization titers (PRNT) were determined against RSV-X and RSV-X-dG, the latter is lacking the G protein. (A-B) Median (± IQR) PRNT against RSV-X were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. (C-D) Median (± IQR) PRNT against RSV-X-dG were compared between healthy and RSV-infected infants as well as between RSV patients with or without oxygen therapy. (E) The PRNT against RSV-X was divided the PRTN against RSV-X-dG, so that values below 1.0 indicate a contribution of anti-G antibodies to the neutralization. The median (± IQR) ratio was compared between plasma from healthy infants and RSV patients with or without oxygen therapy and all were significantly different from 1.0. Statistical analyses employed Mann Whitney <i>U</i> test for comparison between two groups and Kruskall-Wallis for comparison between more than two groups. (*<i>P</i><0.05, <i>**P<</i>0.01).</p

Percent carrying Hi (A), Sa (B), and Mc (C) at 4 time points and density of Sa colonization at baseline and 14 days (D) segregated by success of pneumococcal colonization.

<p>Numbers of subjects carrying each bacteria ranged from 11<b>–</b>17 for <i>H. influenzae</i>, 5<b>–</b>10 for <i>M. catarrhalis</i>, and 13<b>–</b>14 for <i>S. aureus</i>. Asterisk indicates <i>p</i> = 0.008.</p

RSV-specific IgG titer and RSV-IgG avidity do not correlate with disease severity.

<p>RSV-specific IgG levels in plasma of infants was determined by ELISA using virus particles. RSV-IgG avidity was assessed by supplementing NaSCN during ELISA. (A) RSV-specific IgG levels were displayed versus age. (B-C) Median RSV-specific IgG levels (± IQR) were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. (D-E) Median (± IQR) avidity of RSV-IgG in healthy infants was compared to RSV-infected infants or between RSV patients with or without oxygen therapy. Associations were assessed by Spearman correlation test. Statistical analyses employed Mann Whitney U test. (**P<0.01).</p

RSV-specific IgG against individual RSV glycoproteins do not correlate with disease severity.

<p>RSV glycoprotein-specific antibody levels were determined by ELISA against recombinant, soluble ectodomains. Median IgG levels (± IQR) were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. Glycoproteins used for ELISA coating were (A-B) RSV G protein, (C-D) prefusion F protein, and (E-F) postfusion F protein. (G-H) The relative abundance of antibodies against prefusion F compared to G protein as well as pre- compared to postfusion F protein were categorized assessed by subtracting their levels. The median (± IQR) difference was compared between plasma from healthy infants and RSV patients with or without oxygen therapy. No significant differences were observed by Mann Whitney <i>U</i> test for comparison between two groups and Kruskall-Wallis for comparison between more than two groups.</p

RSV-specific IgG against F protein antigenic sites Ø and I not correlate with disease severity.

<p>The abundance of antibodies in human infant plasma that bind to the prefusion F protein antigenic site Ø or the postfusion F protein antigenic site I was determined by competition with site-specific monoclonal antibodies in ELISA. (A-B) Median (± IQR) IgG titer that blocks 25% binding of D25 (site Ø) were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. (C-D) Median (± IQR) IgG titer that blocks 15% binding of 131-2A (site I) were compared between healthy and RSV-infected infants as well as between RSV patients with and without oxygen therapy. No significant differences were observed by Mann Whitney <i>U</i> test.</p

Study population.

<p>Study population.</p