Patient information.

<p>Patient information.</p

RV induced expression of ICAM-1 on BEC is prevented by OM-85 through cAMP and Erk1/2 MAPK.

<p>(A) Increased expression of ICAM-1 by RV and its inhibition by OM-85 pre-incubation (24 hrs). Box-plots represent median and 95% confidence interval of BEC derived from: non-asthma, non-COPD controls (n = 6), asthma (n = 10), and COPD (n = 7). (B) Show the effect of cell signal inhibitors (DDA for cAMP; SB203580 for p38 MAPK and PD98059 for Erk1/2 MAPK) on ICAM-1 expression induced by RV in the presence and absence of OM-85 (10μg/ml). (C) RV and OM-85 induced the secretion of IFN-γ by primary human BEC (n = 5 in each group). (D) OM-85 has a non-significant increasing effect on MyD88 expression by BEC (n = 5 in each group).</p

Dose-dependent preventive effect of OM-85 pre-incubation (2 days) on RV-induced cell death as determined by direct cell counting with Trypan blue exclusion staining.

<p>Box-plots represent median and 95 % confidence intervals for BEC derived from: (A) non-asthma, non-COPD controls (n = 8), (B) asthma (n = 9) and (C) COPD (n = 10). Statistics were performed with paired Student’s t-test, two-sided.</p

OM-85 induced various cell signalling.

<p>(A) Representative Western-blot of the ratio of total and phosphorylated Erk1/2 MAPK and bar chart analysis based on three additional Western-blots in BEC. (B) OM-85 induced activation of intracellular cAMP determined by ELISA in 5 BEC lines. Bars represent mean ± S.E.M. and asterix indicate significant difference compared to non-stimulated BEC. (C) Representative Western-blot of the ratio of total and phosphorylated CREB. Bar chart analysis based on three additional Western-blots in BEC. (D) RV infection was determined by IF and was not affected by inhibition of Erk1/2, p38 MAPK or cAMP (n = 5).</p

Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP - Fig 2

<p>(A) Representative IF images for RV infection (green) in primary BEC; nuclei were stained for cell counting by EVOS live cell staining kit (Thermofisher Scientific). Light microscopic images are depicted for the effect of OM-85 and RV infection on BEC phenotype. (B-D) Quantitation of RV infection of BEC by IF. BEC were pretreated for 24 or 48 hrs with OM-85 (10 μg/ml). Bars represent mean±SEM of RV positive BEC derived from: non-asthma, non-COPD controls (n = 8), (C) COPD (n = 10) and (D) asthma (n = 9). Statistics were performed with paired Student’s t-test, two-sided.</p

Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes

<p><i>Purpose</i>: The mechanisms involved in the reported beneficial effects of Calcium dobesilate monohydrate (CaD) for the treatment of diabetic retinopathy (DR) remain to be elucidated. The main aim of the present study is to examine whether CaD prevents early events in the pathogenesis of DR such as neurodegeneration and vascular leakage. In addition, putative mediators of both neurodegeneration (glutamate/GLAST, ET-1/ETB receptor) and early microvascular impairment (ET-1/ETA receptor, oxidative stress, VEGF, and the PKC-delta-p38 MAPK pathway) have been examined.</p> <p><i>Methods</i>: Diabetic (db/db) mice were randomly assigned to daily oral treatment with CaD (200 mg/Kg/day) (<i>n</i> = 12) or vehicle (<i>n</i> = 12) for 14 days. In addition, 12 non-diabetic (db/+) mice matched by age were used as the control group. Functional abnormalities were assessed by electroretinography. Neurodegeneration and microvascular abnormalities were evaluated by immunohistochemistry and Western blot. Glutamate was determined by HPLC.</p> <p><i>Results</i>: CaD significantly decreased glial activation and apoptosis and produced a significant improvement in the electroretinogram parameters. Mechanistically, CaD prevented the diabetes-induced up-regulation of ET-1 and its cognate receptors (ETA-R and ETB-R), which are involved in microvascular impairment and neurodegeneration, respectively. In addition, treatment with CaD downregulated GLAST, the main glutamate transporter, and accordingly prevented the increase in glutamate. Finally, CaD prevented oxidative stress, and the upregulation of VEGF and PKC delta-p38 MAPK pathway induced by diabetes, thus resulting in a significant reduction in vascular leakage.</p> <p><i>Conclusions</i>: Our findings demonstrate for the first time that CaD exerts neuroprotection in an experimental model of DR. In addition, we provide first evidence that CaD prevents the overexpression of ET-1 and its receptors in the diabetic retina. These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of CaD in very early stages of DR.</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 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

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

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