Frequency of <i>HLA-DRB1</i> genotypes obtained using SSO (x-axis) and CAPSeq (y-axis) compared at 4-digit resolution.

<p>The dashed line represents the theoretical identity between the two methods. Pearson’s correlation coefficient (r) exceeded 0.999.</p

The Clustering and Alignment of Polymorphic Sequences (CAPSeq) software application illustrated as a schematic.

<p><i>Input Data:</i> Next-generation sequence data formatted as modified FASTQ files consisting of sequences and corresponding Q-scores along with an additional input data file containing known <i>HLA</i> allele sequences. <i>CAPSeq Application:</i> The analysis software can be broken down into 3 principle steps consisting of those developed to align sequences and use corresponding Q-scores to generate a weighted pairwise similarity score (step 1) that can be analyzed via diffusion mapping, followed by K-means clustering to enable the identification of homogeneous sequence groups (step 2) followed by Bootstrap Aggregating, Bagging, of multiple analyses of the data to ensure genotyping precision (step 3). <i>Output Data:</i> The <i>HLA</i> genotyping data is provided as a tab delimited text file containing the most likely allelic match between the CAPSeq generated consensus sequences and list of known <i>HLA</i> alleles.</p

Comparison of SSO based 4-digit <i>HLA-DRB1</i> genotyping with CAPSeq.

<p>Comparison of SSO based 4-digit <i>HLA-DRB1</i> genotyping with CAPSeq.</p

<i>HLA-DRB1</i> alleles.

<p>Allele frequencies and rankings are taken from Maiers et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059835#pone.0059835-Maiers1" target="_blank">[19]</a> for European <i>-DRB1</i> and from Lancaster et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059835#pone.0059835-Lancaster1" target="_blank">[20]</a> for worldwide frequencies. UC cohort frequencies are determined from CAPSeq genotyping results.</p

Image_14_Co-Stimulation-Impaired Bone Marrow-Derived Dendritic Cells Prevent Dextran Sodium Sulfate-Induced Colitis in Mice.TIF

<p>Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7–10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.</p

Image_11_Co-Stimulation-Impaired Bone Marrow-Derived Dendritic Cells Prevent Dextran Sodium Sulfate-Induced Colitis in Mice.TIF

<p>Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7–10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.</p

Image_15_Co-Stimulation-Impaired Bone Marrow-Derived Dendritic Cells Prevent Dextran Sodium Sulfate-Induced Colitis in Mice.TIF

<p>Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7–10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.</p

Image_13_Co-Stimulation-Impaired Bone Marrow-Derived Dendritic Cells Prevent Dextran Sodium Sulfate-Induced Colitis in Mice.TIF

<p>Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7–10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.</p

HGF modulates IL-6 production in LPS stimulated macrophages.

<p>BMM derived from C57BL6 mice were pretreated with or without 10 pg and 10 ng HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of the mean (± SEM) of three independent experiment done in triplicate, * indicates <0.001.</p

A MET kinase inhibitor abrogates HGF suppression of IL-6.

<p>Using an optimal dose of the MET inhibitor SU11274 (1 µg), BMM were pretreated for 2 hours before an overnight incubation with 10 and 100 pg HGF and 24 hour stimulation with LPS. Results are representative of the mean (± SEM) of three independent experiments done in triplicate. *, p = 0.02 vs. the respective control group.</p