Chimeric anti-SEB 82M and lovastain provide <i>in vivo</i> protection against SEB toxicity in HLA-DR3 transgenic mice.

<p>(<b>A</b>) Inhibition of SEB-mediated hypothermic effect. Age-matched HLA-DR3 transgenic mice were injected with the following: Ch 82 M+Lova+SEB (♦), 1 mg chimeric 82 M+1 mg lovastatin+50 µg SEB; Ch 82 M+SEB (▪), 1 mg chimeric 82 M+50 µg SEB; Lova+SEB (▴), 1 mg lovastatin +50 µg SEB; SEB alone (×), 50 µg SEB in PBS. Rectal temperatures were recorded at the indicated time points. Error bars are the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>B</b>–<b>D</b>) Inhibition of SEB-induced cytokine production. Serum levels of interleukin-2 (<b>B</b>), interferon-γ (<b>C</b>) and interleukin-6 (<b>D</b>) were determined at 6 hours post-SEB injection in groups of age-matched HLA-DR3 transgenic mice treated with the following: naïve, PBS only; Ch 82 M + SEB, 1 mg Ch 82 M+50 µg SEB; Lova + SEB, 1 mg lovastatin +50 µg SEB; Ch 82 M + Lova + SEB, 1 mg Ch 82 M+1 mg lovastatin+50 µg SEB; SEB alone, 50 µg SEB in PBS. Differences between the combination of Ch 82 M and lovastatin and either drug or antibody alone were significant (P<0.05) in inhibiting SEB-induced cytokine production. Each bar represents the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>E</b>) Protection against SEB-mediated death. Survival was monitored within groups of age-matched HLA-DR3 transgenic mice receiving the following: SEB alone, 50 µg SEB in PBS; Ch 82+SEB, 1 mg of Ch 82 M+50 µg SEB; Lova+SEB, 1 mg of lovastatin+50 µg SEB; Ch 82 M+Lova + SEB, 1 mg Ch 82 M+1 mg lovastatin+50 µg SEB. Survival was monitored for 7 days. Combination of Ch 82M and lovastatin provided statistically significant protection (P<0.01) against SEB-induced death compared with mice treated with SEB alone. The data shown are representative of two or more independent experiments.</p

Chimeric anti-SEB 82 M and 63 synergistically protect HLA-DR3 mice from the toxic effects of SEB.

<p>(<b>A</b>) Protection against SEB-induced hypothermia. Age-matched HLA-DR3 transgenic mice were injected with the following: Ch 82 M+Ch 63+SEB (♦), 500 µg chimeric 82 M+500 µg chimeric 63+50 µg SEB; Ch 63+SEB (▪), 1 mg chimeric 63+50 µg SEB; Ch 82 M+SEB (▴),1 mg chimeric 82 M+50 µg SEB; Hu IgG1κ+SEB (×), 1 mg human IgG1κ+50 µg SEB; SEB alone (*); 50 µg SEB in PBS. Rectal temperatures were recorded at the indicated time points. Error bars are the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. <b>(B</b>–<b>D)</b> Inhibition of SEB-induced cytokine production. Serum levels of interleukin-2 (<b>B</b>), interferon-γ (<b>C</b>), and interleukin-6 (<b>D</b>) were determined at 6 hours post-SEB injection in groups of age-matched HLA-DR3 transgenic mice treated with the following: naïve, PBS only; Ch 82 M+SEB, 1 mg Ch 82 M+50 µg SEB; Ch 63+SEB, 1 mg Ch 63+50 µg SEB; Ch 82 M+Ch 63+SEB, 500 µg Ch 82 M+500 µg Ch 63+50 µg SEB; SEB alone, 50 µg of SEB in PBS. Differences between the combination of anti-SEBs and either antibody used alone were significant (P<0.05) in inhibiting SEB-induced cytokine production. Each bar represents the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>E</b>) Appearance of mice protected with anti-SEB and unprotected mice 6 hours after a 50 µg dose of SEB. The mouse on the left received no protective antibody, suffered hyperthermia, shivered and displayed hunched posture and a rough coat. The mice on the right, which were treated with 50 µg SEB+500 µg Ch 82 M+500 µg Ch 63 appeared normal, were sleek of coat and animated. (<b>F</b>) Protection against SEB-mediated death. Survival was monitored within groups of age-matched HLA-DR3 transgenic mice receiving the following: SEB alone, 50 µg SEB in PBS; Hu IgG1κ+SEB, 1 mg human IgG1κ+50 µg SEB; Ch 82 M+SEB, 1 mg Ch 82 M+50 µg SEB; Ch 63+SEB, 1 mg Ch 63+50 µg SEB; Ch 82 M+Ch 63+SEB, 500 µg Ch 82 M+500 µg Ch 63+50 µg SEB. Combination of Ch 82 M and Ch 63 provided statistically significant protection (P<0.001) against SEB-induced death compared with untreated controls. The data shown are representative of two or more independent experiments.</p

Synergistic neutralization /inhibition of SEB-mediated T-cell activation by a combination of anti-SEB antibody and lovastatin.

<p>(<b>A</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in BALB/c splenocytes. BALB/c splenocytes were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 100 ng/ml SEB; Hu IgG1κ+SEB, 10 µg/ml Human IgG1κ+100 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+100 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+100 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg /ml chimeric anti-SEB 82 M+2.5 µM lovastatin+100 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M+lovastatin was significantly more inhibitory than either agent alone (P<0.01). (<b>B</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in HLA-DR3 transgenic mice splenocytes. HLA-DR3 transgenic mice splenocytes were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 10 ng/ml SEB; HuIgG1κ+SEB, 10 µg/ml Human IgG1κ+10 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+10 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+10 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg/ml chimeric anti-SEB 82 M+2.5 µM lovastatin+10 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M and lovastatin was significantly more inhibitory than either agent alone (P<0.01). (<b>C</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in human PBMCs. PBMCs were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 1 ng/ml SEB; HuIgG1κ+SEB, 10 µg/ml Human IgG1κ+1 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+1 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+1 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg/ml chimeric anti-SEB 82 M+2.5 µM lovastatin+1 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M and lovastatin was significantly more inhibitory than either agent alone (P<0.01).</p

Novel GOS-Only Clusters Are More Interconnected Than a Size-Matched Sample of Clusters

<p>Red line, novel clusters; green line, size-matched sample; blue line (right axis), log₂ ratio of fraction novel clusters recovered divided by fraction sample clusters recovered.</p

Enrichment in the GOS-Only Set of Clusters for Viral Neighbors

<p>Cluster sets from left to right are: I, GOS-only clusters with detectable BLAST, HMM, or profile-profile homology (Group I); II, GOS-only clusters with no detectable homology (Group II); I-S, a sample from all clusters chosen to have the same size distribution as Group I; II-S, a sample from all clusters chosen to have the same size distribution as Group II; I-V, a subset of clusters in Group I containing sequences collected from the viral size fraction; II-V, a subset of clusters in Group II from the viral size fraction; and all clusters. Notice that although predominantly bacterial, GOS-only clusters are assigned as viral based on their neighbors more often than the size-matched samples and the set of all clusters.</p

GOS-Only Clusters Are Enriched for Sequences of Viral Origin Independently of the Kingdom Assignment Method Employed

<p>For each panel, clusters are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050016#pbio-0050016-g004" target="_blank">Figure 4</a>. For (A–C), a kingdom is assigned to each neighboring ORF within each cluster set; the percentage of all neighboring ORFs with a given kingdom assignment is plotted. For (D–F), a kingdom is assigned to each cluster if more than 50% of all that cluster's neighbors with a kingdom assignment share the same assignment; the percentage of clusters in each set with a given assignment is plotted. In (A) and (D), a kingdom is assigned to a neighboring ORF by a majority vote of the top four BLAST matches to a protein in NCBI-nr (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050016#s3" target="_blank">Materials and Methods</a>). In (B) and (E), a kingdom is assigned if all eight highest-scoring BLAST matches agree in kingdom. In (C) and (F), all ORFs on a scaffold are assigned the same kingdom by voting among all ORFs with BLAST matches to NCBI-nr on that scaffold (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050016#s3" target="_blank">Materials and Methods</a>). In all graphs, only clusters with at least one assignable neighbor are considered. When compared to the size-matched controls, in all cases the GOS-only clusters show enrichment for viral sequences.</p

Coverage of GOS-100 and Public-100 by Pfam and Relative Sizes of Pfam Families by Kingdom, Sorted by Size

<p>The public-100 sequences are annotated using the NCBI taxonomy and the source public database annotations. GOS-100 sequences were given kingdom weights as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050016#s3" target="_blank">Materials and Methods</a>. For each kingdom, the fraction of sequences with ≥1 Pfam match are shown, while the ten largest Pfam families shown as discrete sections whose size is proportional to the number of matches between that family and GOS-100 or public-100 sequences. Pfam families that are smaller than the ten largest are binned together in each column's bottom section. Pfam covers public-100 better than GOS-100 in all kingdoms, with the greatest difference occurring in the viral kingdom, where 89.1% of public-100 viral sequences match a Pfam domain, while only 27.5% of GOS-100s have a sequence match.</p

Content of Protease Types in NCBI-nr and GOS, and Kingdom Distribution of All Proteases

<p>Due to the highly redundant nature of some NCBI-nr protease groups, nonredundant sets for both NCBI-nr and GOS are computed; these nonredundant sets are referred to as NCBI-nr60 and GOS60.</p

Structure and GOS Homologs of Hypothetical Protein AF1548

<p>Yellow bars represent β-strands. Highlighted are predicted catalytic residues: 38D, 51E, and 53K.</p

Venn Diagram Showing Breakdown of the 17,067 Medium and Large Clusters by Three Categories—GOS, Known Prokaryotic, and Known Nonprokaryotic

<p>Venn Diagram Showing Breakdown of the 17,067 Medium and Large Clusters by Three Categories—GOS, Known Prokaryotic, and Known Nonprokaryotic</p