Journal: PLoS medicine

Article Title: In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Figure 3. Antibodies against the Celiac Peptide Bind the Self-Antigens MPMR2 and TLR4 (A) Sera of patients with active CD contain IgA antibodies directed against MPMR2; such reactivity is not present in patients on GFD. A K562 cell lysate was probed with rabbit antiserum raised against a peptide (VEKIGGASSRGE) of the MPMR2 (Lane 1), with antibodies affinity-purified against the celiac peptide (lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from patients with active disease on GCD (Lanes 4, 6, and 8), and with sera from the same patients on GFD (Lanes 5, 7, and 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection. (B) Cell lysate from untransfected 293T cells was probed with the monoclonal antibody against TLR4 (Lane 1). Cell lysate from 293T cells transfected with the human TLR4 gene was probed with a monoclonal antibody directed against TLR4 (Lane 2), and with antibodies affinity- purified against the celiac peptide (Lane 3). Cell lysate from 293T cells transfected with the TLR4 gene was probed with biotin-labelled anti- TLR4 monoclonal antibody (Lane 4). Cell lysate from 293T cells transfected with the TLR4 gene was probed first with affinity-purified anti-peptide antibodies, followed by an incubation with biotin-labelled anti-TLR4 monoclonal antibody (Lane 5). Cell lysate from human plasmocytoid dendritic cells was probed with the monoclonal antibody against TLR4 (Lane 6). Cell lysate from human monocytes was probed with the monoclonal antibody against TLR4 (Lane 7) and with affinity- purified anti-peptide antibodies (Lane 8). (C) Inhibition of binding of anti-celiac peptide antibodies to solid-phase TLR4 peptide by liquid-phase TLR4 peptide (blue line), by celiac peptide (red line), and VP-7 peptide (red line), but not by an irrelevant control peptide (green line). The y-axis represents percentage of inhibition, and the x-axis indicates inhibitor concentration (lg/ml). DOI: 10.1371/journal.pmed.0030358.g003

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Affinity Purification, Control, Transfection, Incubation, Inhibition, Binding Assay, Concentration Assay

Journal: PLoS medicine

Article Title: In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Figure 6. Pro-Inflammatory Cytokines Produced by Activated Monocytes Levels of IL-6 (A), IL-12 (B), and TNF-alpha (C) released in the supernatant by monocytes incubated with medium alone (Line 1), with antibodies directed against an irrelevant peptide (Line 2), with LPS (Line 3), with pooled Igs isolated from the 22 patients with active CD (Line 4), with pooled Igs isolated from the same patients on GFD (Line 5), with purified anti-celiac peptide antibodies obtained from ten patients (line 6), with purified anti-celiac peptide antibodies in the presence of an irrelevant mouse IgG2b antibody (20 lg/ml) (Line 7), and with purified anti-celiac peptide antibodies in the presence of the neutralizing mouse mono- clonal antibody anti-TLR4, clone HTA 125 (20 lg/ml) (Line 8). The y-axis represents the cytokine concentration expressed as pg/ml. Data represent the mean 6SD of three independently performed experi- ments. DOI: 10.1371/journal.pmed.0030358.g006

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Produced, Incubation, Isolation, Purification, Concentration Assay

Journal: PLoS medicine

Article Title: In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Figure 7. Anti-Celiac Peptide Antibodies Activate TLR4 in Cells Trans- fected with the TLR4 Gene Activation of NF-jB upon engagement of TLR4. (A) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4); by affinity-purified anti-celiac peptide antibodies, 4 lg/ml (5), 2 lg/ml (6), 1 lg/ml (7), 0.5 lg/ml (8) by affinity-purified antibodies directed against an irrelevant control peptide, 4 lg/ml (9), 2 lg/ml (10), 1 lg/ml (11), and 0.5 lg/ml (12). (B) Stimulation of 293T cells transfected with TLR4 by LPS 100 ng/ml (1), 10 ng/ml (2), 1 ng/ml (3), 0.1 ng/ml (4), by affinity-purified antibodies against an irrelevant peptide 1 lg/ml (5), by affinity-purified anti-celiac peptide antibodies 1 lg/ml (6), by affinity-purified anti-celiac peptide antibodies 1 lg/ml in the presence of 1 lg/ml anti-TLR4 monoclonal antibody (7), by affinity-purified anti-celiac peptide antibodies 1 lg/ml in the presence of 1 lg/ml celiac peptide (8), by affinity-purified anti-celiac peptide antibodies 1 lg/ml in the presence of 1 lg/ml irrelevant control peptide (9), by affinity-purified anti-celiac peptide antibodies 1 lg/ml in the presence of 1 lg/ml recombinant human tTG (10), by affinity-purified anti-VP-7 peptide antibodies 1 lg/ml (11), by affinity-purified anti-VP-7 peptide antibodies 1 lg/ml in the presence of 1 lg/ml VP-7 peptide (12), by affinity-purified anti-VP-7 peptide antibodies 1 lg/ml in the presence of 1 lg/ml celiac peptide (13), and by affinity-purified anti-celiac peptide antibodies 1 lg/ml in the presence of 1 lg/ml ovalbumin (14). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used). DOI: 10.1371/journal.pmed.0030358.g007

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Activation Assay, Transfection, Affinity Purification, Control, Recombinant, Positive Control, Concentration Assay

Journal: PLoS medicine

Article Title: In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Figure 8. Antibodies against the Celiac Peptide Recognize Desmoglein 1 (A) Sequence homology between the celiac peptide and cell junction proteins. The peptide sequence was compared with known protein sequences using the BLASTP via the NCBI BLAST network service (colons indicate identity and asterisks indicate conservative substitutions). (B) Sera of patients with active CD contain IgA antibodies directed against desmoglein 1; such reactivity is not present in patients on GFD. Recombinant desmoglein 1 was probed with antibodies affinity purified against the celiac peptide (Lane 1), with antibodies affinity purified against an irrelevant control peptide (Lane 2), with sera from patients with active disease on GCD (Lanes 3 and 5), and with sera from the same patients on GFD (Lanes 4 and 6). Biotin-labelled primary antibodies followed by peroxidase-labelled avidin (Lanes 1 and 2) and an anti- human IgA antibody (Lanes 3–6) were used for detection. (C) The binding of affinity-purified anti-celiac peptide antibodies to solid- phase desmoglein 1 is inhibited by desmoglein 1 (blue line), tTG (red line), celiac peptide (purple line), VP-7 peptide (yellow line), and TLR4 peptide (green line), but not by the irrelevant control peptide (orange line). The y-axis represents percentage of inhibition, and the x-axis indicates inhibitor concentration (lg/ml). DOI: 10.1371/journal.pmed.0030358.g008

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Sequencing, Recombinant, Affinity Purification, Control, Avidin-Biotin Assay, Binding Assay, Inhibition, Concentration Assay

Journal: PLoS medicine

Article Title: In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

doi: 10.1371/journal.pmed.0030358

Figure Lengend Snippet: Figure 10. Features of Rabbit Anti-VP-7 Antibodies (A) Binding of rabbit anti-VP-7 antibodies to tTG (blue line). Red line indicates pre-immune rabbit serum. (B) Binding of rabbit anti-VP-7 antibodies to VP-7 peptide (blue line), celiac peptide (red line), desmoglein peptide (yellow line), and TLR4-peptide (green line). Purple line indicates binding to the irrelevant control peptide. (C) TLR4 activation by LPS (100 ng/ml) (1), affinity-purified human anti-celiac peptide antibodies (2), rabbit anti-VP-7 antibodies (3), and pre-immune rabbit serum (4). Results are expressed as percentage of positive control, where the positive control is the OD value obtained upon stimulation of TLR4 transfected cells with 100 ng/ml LPS (maximal concentration used). (D) Confluent T84 monolayers were treated for 3 h with control normal human Ig (1), affinity-purified human antibodies to the irrelevant control peptide (2), affinity-purified human anti-celiac peptide antibodies (3), rabbit anti-VP-7 antibodies (4), and pre-immune rabbit serum (5). DOI: 10.1371/journal.pmed.0030358.g010

Article Snippet: The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex), was used for inhibition experiments.

Techniques: Binding Assay, Control, Activation Assay, Affinity Purification, Positive Control, Transfection, Concentration Assay