Surface Plasmon Resonance analysis.

<p>SPR experiment showing binding of hCXCR3 and hCCR6 peptides to bispecific stabilized antibody and to control parental humanized IgG’s. GLM chip containing immobilized Protein G’ docked in ProteOn XPR3 instrument was utilized for this experiment. BsAb, hX3 and hR6 IgG1 were captured onto protein G’ chip surface via their Fc regions. hCXCR3 peptide (Panels A, B, C), hCCR6 peptide (Panels D, E, F) and a mixture of hCCR6 and hCXCR3 peptides (G,H,I) were injected over immobilized bispecific Ab (top panels, black curves), hR6 IgG1 (middle panels, blue curves) and hX3 (bottom panels, red curves).</p

Antibody formats used in this study.

<p>Ribbon diagram representation of mX3 and mR6 Fv structure (black ribbons) superimposed with hX3 (A) (Blue ribbon) and hR6 (B) (green ribbon). The CDRs are shown in red. Schematic illustration of the vectors used for (C) scFv; (D) humanized IgG1; (E) Bispecific (scFv)₄ IgG-like and (F) Bispecific (scFv)₂ IgG-like expression. Abbreviations: LacZ, promoter of the bacterial Lac operon; pelB and SP, signal sequence for the secretion of the recombinant antibodies; L, (G₄S)₃ peptide linker; H, 6-HIS Tag; CMV, human cytomegalovirus promoter; pA, polyadenylation site. Schematic representation of the recombinant antibody formats used in this study. (G) humanized scFv; (H) humanized IgG1; (I) humanized (scFv)₄ IgG-like bispecific antibody; (J) non stabilized humanized (scFv)₂ IgG-like bispecific antibody and (K) stabilized humanized (scFv)₂ IgG-like bispecific antibody.</p

Flow cytometric analysis.

<p>(A) Reactivity of mAb and BsAb on hCXCR3 and hCCR6 L1.2 expressing cells. The hCXCR3 and hCCR6 L1.2 expressing cells were mixed and incubated with an isotype control antibody (grey line) or mX3 mAb IgG1 (blue line; top left panel); hX3 IgG1 (blue line; top middle panel); hX3 scFv (blue line; top right panel); mR6 IgG1 (green line; bottom left panel); hR6 IgG1 (green line; bottom middle panel) or BsAb (purple line; bottom right panel). The murine IgG1 were stained with an anti-mouse igG Fc specific PE-conjugated; the humanized scFv was stained with an anti-6HIS antibody followed by an anti-mouse igG Fc specific PE-conjugated antibody; the humanized IgG1 and BsAb were stained with an anti-human kappa PE conjugated antibody. (B) Reactivity of mAb and BsAb on purified human lymphocytes. Human lymphocytes were isolated from blood on a ficoll gradient and stained with an anti-human CD3-BV650 and an anti-human CD4 Pacific Blue conjugated antibody. The lymphocytes in the CD3+/CD4+ gate were stained with isotype controls (left panels); FITC conjugated hR6 mAb and hX3 (detected with anti-human Kappa PE-conjugated antibody) (middle panel) or BsAb (right panel) followed by an anti-human Kappa PE-conjugated antibody. (C) Intracellular staining for FoxP3 protein in lymphocytes isolated from human blood. Activated human lymphocytes were gated on CD3+/CD4+/hX3+ (left panel); CD3+/CD4+/hR6+ (left panel); CD3+/CD4+/BsAb+ (left panel) and stained intracellularly for FoxP3. (D) Intracellular staining for IFN-γ and IL-17A proteins in PMA/ionomycin-stimulated lymphocytes isolated from human blood. Activated human lymphocytes were gated on CD3+/CD4+/hX3+ (left panel); CD3+/CD4+/hR6+ (left panel); CD3+/CD4+/BsAb+ (left panel) and stained intracellularly for IFN-γ and IL-17A.</p

In vitro characterization of mAbs functions.

<p>Chemotaxis inhibition by hX3; hR6 and BsAb. (A) The hCCR6 and hCXCR3 L1.2 cells were mixed; preincubated with an Isotype control and allowed to migrated towards a combination of hIP-10 and hCCL20 chemokines in the lower chamber. The hCCR6 and hCXCR3 L1.2 cells chemotaxis to hCCL20 and hIP-10 was carried out in the presence of increasing concentrations of purified R6; X3 and BsAb. Statistical analyses were performed using a t-test comparison between Isotype and antibodies groups (* p<0.05, ** p<0.01). (B) Cytotoxicity of activated human NK cells (effector) against a mixture of hCCR6 and hCXCR3 L1.2 cells (target) by R6; X3; R3 + X3 and BsAb <i>in vitro</i>.</p

Purification of mAbs and BsAb.

<p>SDS-PAGE analysis of the various affinity purified antibodies under non-reducing conditions (A) and reducing conditions (B). The molecular weight (kDa) are shown on the left. Screening of monoclonal CHO-DG44 cells expressing the BsAb with ClonePix FL. The transfected cells were plated in a semi-solid media and visualized under bright light (C) and fluorescence (D). Size Exclusion Chromatography profile of non stabilized (dashed line) and stabilized (black line) BsAb. Arrows indicate the molecular weight of the standards.</p

Deficiency of dietary fiber modulates gut microbiota composition, neutrophil recruitment and worsens experimental colitis

Ulcerative colitis is an inflammatory disease of the colon that is associated with colonic neutrophil accumulation. Recent evidence indicates that diet alters the composition of the gut microbiota and influences host–pathogen interactions. Specifically, bacterial fermentation of dietary fiber produces metabolites called short-chain fatty acids (SCFAs), which have been shown to protect against various inflammatory diseases. However, the effect of fiber deficiency on the key initial steps of inflammation, such as leukocyte–endothelial cell interactions, is unknown. Moreover, the impact of fiber deficiency on neutrophil recruitment under basal conditions and during inflammation in vivo is unknown. Herein, we hypothesized that a fiber-deficient diet promotes an inflammatory state in the colon at baseline and predisposes the host to more severe colitis pathology. Mice fed a no-fiber diet for 14 days showed significant changes in the gut microbiota and exhibited increased neutrophil-endothelial interactions in the colonic microvasculature. Although mice fed a no-fiber diet alone did not have observable colitis-associated symptoms, these animals were highly susceptible to low dose (0.5%) dextran sodium sulphate (DSS)-induced model of colitis. Supplementation of the most abundant SCFA, acetate, prevented no-fiber diet-mediated enrichment of colonic neutrophils and colitis pathology. Therefore, dietary fiber, possibly through the actions of acetate, plays an important role in regulating neutrophil recruitment and host protection against inflammatory colonic damage in an experimental model of colitis