Activation of PBMC and MoDC from COPD patients and healthy subjects by OM-85.

<p><b>A</b>) PBMC and <b>B</b>) MoDC (both 10⁶/ml) were stimulated as indicated in the presence or absence of 500 U/ml IFNγ or 100 ng/ml TNF-α. After 24 hours, supernatants were collected and analyzed by ELISA. Figure shows the results of healthy donors (open histograms) compared to COPD patients (black histograms). *P<0.05 by paired Student's <i>t</i> test.</p

Induction of selected cytokines and chemokines by OM-85 in MoDC.

<p><b>A</b>) MoDC (10⁶/ml) were stimulated with OM-85 as indicated and with 100 ng/ml LPS (IL-6, BAFF, CCL2, CXCL8 and CXCL6) or 10 ng/ml LPS (CCL3, CCL20 and CCL22) as a positive control. After 24 hours, supernatants were collected and analyzed by ELISA. Ut = untreated. *P<0.05 and **P<0.01 by Dunnett's Multiple Comparison Test. <b>B</b>) Supernatants of MoDC stimulated with OM-85 induce a G-protein-dependent migration of PMN. As a comparison, migration of PMN was elicited with unstimulated supernatants+CXCL8 and PMA. As expected, migration to CXCL8 was inhibited by both 10 nM M3 and 750 ng/ml, <i>Pertussis toxin</i> (P.Tox) while migration to PMA was not. Results are expressed as chemotactic index over migration to supernatants of unstimulated MoDC and represent means+/−SD of three independent Boyden chamber experiments. *P value<0.05 and ** P value<0.01 by Dunnett's Multiple Comparison Test.</p

Activation of the NF-kB and MAPK pathways by OM-85 in MoDC.

<p><b>A</b>) Immature human MoDC were stimulated with 100 µg/ml OM-85 for 30, 60, 90 and 120 minutes. 100 ng/ml LPS was used as a positive control. After cell lysis and protein fractionation, cytoplasmic (Cyto) and nuclear (Nuclei) extracts were blotted against NF-kB p65 and IkBα. β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of eight. <b>B</b>) EMSA (upper panel) and supershift (lower panel) showing the induction of NFkBp65-DNA binding activity by OM-85 in human moDC stimulated as in A). Signal specificity was assessed by competing each sample with a 125-fold excess unlabeled probe (lanes 2,4,6,8,10 upper panel). The image depicts results obtained in one representative donor out of four. <b>C</b>) OM-85 induces the production of luciferase in THP1 cells bearing a NF-kB-reporter plasmid (NF-kB pGL4, striped histograms). THP1 cells were stimulated with 1 µg/ml LPS and 1000 µg/ml OM-85. As expected, THP1 untransfected cells (untransfected, empty histograms) did not produce luciferase in response to stimulation. Similar results were obtained when cells were transfected with the pGL4 empty backbone (pGL4, black histograms). Results are expressed as mean+/−SD of three independent experiments. *P value<0.01 by Dunnett's Multiple Comparison Test. <b>D</b>) OM-85 activates the MAPK pathway. Cell extracts prepared as in A) were blotted with antibodies specific for phophorilated ERK1/2 (Cyto, upper panel) and total ATF2 and c-Jun (Nuclei, lower panel). β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of three. <b>E</b>) OM-85 induces NF-kB- and MAPK-dependent gene transcription. Immature MoDC were stimulated with 100 µg/ml OM-85 (open circles) and 100 ng/ml LPS (black circles) for 2, 4, 8 and 24 hours. After RNA extraction, reverse transcription and DNAse I digestion, samples were amplified by Q-PCR using gene-specific primers. Results represent means+/−SE of three independent donors and are expressed as fold induction (FI) over unstimulated samples (0).</p

Characteristic of COPD patients enrolled in the study.

<p>Table reports the main features (age, sex, age at diagnosis of COPD, disease stadium, therapy and notes) of selected patients <b>(P).</b></p

Synergism between OM-85 and pro-inflammatory agonists in MoDC.

<p>MoDC (10⁶/ml) were stimulated as indicated. After 24 hours, supernatants were collected and analyzed by ELISA. *P<0.05 and **P<0.005 by paired Student's <i>t</i> test.</p

Activation of primary DC subsets by OM-85.

<p><b>A</b>) MDC and <b>B</b>) PDC were isolated from buffy coats of three independent healthy donors and stimulated as indicated (10⁶/ml). After 24 hours, supernatants were collected and analyzed by ELISA. *P<0.05 by Dunnett's Multiple Comparison Test.</p

Intradermal injection of Activin A induces the differentiation of dermal and epidermal Langerhans cells in human skin explants.

<p>Langerin expression was evaluated in the epidermis and dermis (full thickness skin explants) of skin explants, untreated and 72 hrs after i.d. injection of medium or 100 ng Activin A (magnification 100X, inset 400X) The number of Langerin⁺ cells were quantified in skin explants by evaluating six different skin sections (0.05 mm²/field; means±SD). * p<0.05 by Student's t test vs. medium (lower right panel).</p

Dermal accumulation of Langerhans cells in lichen planus is associated to abundant production of Activin A.

<p>Sections from normal skin (NS) (<i>a</i> and <i>d</i>) and lichen planus (LP) (<i>b, c, e, f</i>) biopsies were stained for Langerin (<i>a</i>–<i>c</i>) and Activin A (<i>d–f</i>). In normal skin, Langerin⁺ cells are regularly distributed in basal and suprabasal layers and show multiple fine dendrites; no positive cells are detectable in the dermis (panel <i>a</i>). In LP biopsies, in addition to intraepidermal LC, accumulation of Langerin⁺ cells is observed in the dermis within the dense monuclear cell infiltrate (panel <i>b</i>). At high power view, Langerin⁺ cells show an ovoidal/dendritic shape (panel <i>c</i>) and are found surrounding Factor VIII⁺ dermal blood vessels (arrow head, inset in <i>c</i>). Serial sections from the same tissue blocks were stained for Activin A. Normal skin (panel <i>d</i>) showed weak intraepithelial reactivity (red arrow head); in the dermis, mast cells and occasional spindle cells were positive for Activin A (black arrow heads). In LP, Activin A was strongly induced in the superficial layers of epidermis; in the dermis, a diffuse reactivity can be observed in numerous cells within the inflammatory infiltrate (panel <i>e</i>). This cell population includes endothelial cells and a mixture of non-lymphoid mononuclear cells (panel <i>f</i>). Magnification 100x (<i>a, b, d, e</i>; scale bar 200 micron) and 400x (<i>c, f</i>; scale bar 50 micron).</p

Activin A induces Langerhans cells differentiation in epidermis-depleted skin explants.

<p>Langerin expression was evaluated in the dermal layer, separated from skin explants by dispase digestion and subsequently treated for 72 hrs after i.d. injection with 100 ng Activin A (magnification 100X, inset 400X.).</p

Activin A promotes Langerhans cell differentiation from human CD14⁺ monocytes.

<p>(A) Phenotypic analysis of monocytes cultured for 6 days with GM-CSF and IL-4 in the presence of Activin A (Act A-LC) or TGFβ1 (TGFβ1-LC). Cells were stained with the indicated moAbs (filled histograms) or isotype-matched negative control moAbs (open histograms). Percentages of positive cells are shown in the upper right corner of each histogram. The figure shows one experiment representative of at least five independent cultures. (B) Electron microscopy analysis of Act A-LC. Act A-LC exhibited abundant dendritic membrane protrusions and lobulated or indented nuclei (left panel, 3,000X, bar 40 µm). Cytoplasm presented a rough endoplasmic reticulum, many multilamellar organelles and numerous electron-dense structures reminiscent of Birbeck granules (right panel, 12,000X, bar 1 µm). The inset shows rod-shaped Birbeck granules (200,000X, bar 20 µm). (C) TGFβ1 and Activin A mRNA expression in Act A-LC and TGFβ1-LC cultures. Monocytes were cultured in the presence of Act A or TGFβ1 for the indicated time and the expression of TGFβ1 and Activin A mRNA was determined by real-time PCR, relative to GAPDH mRNA used as internal control. The expression level in freshly isolated monocytes was assumed as the 1.0 value. Similar results were obtained in three different donors. (D) Effects of different TGF family members on LC differentiation. Monocytes were cultured for 6 days with GM-CSF in the presence of 10 ng/ml TGFβ1, 100 ng/ml Activin A, or 100 ng/ml BMP6 and analyzed for Langerin, E-caderin and CCR6 expression by flow cytometry analysis. Data are representative of at least four independent cultures.</p