Abstract Early non-invasive detection of lung cancer is a precondition for enabling better prognosis supported by new innovative therapy regimes. The aim of our study was to evaluate angiogenic and inflammatory proteins in exhaled breath condensate (EBC) as markers for lung cancer. Our report presents a diagnostic study of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α) in EBC of 300 individuals, 84 patients with lung cancer, 111 patients with stable chronic obstructive pulmonary disease (COPD), and in 105 healthy controls. Detection of VEGF and bFGF in EBC was applicable to discriminate cancer patients from COPD patients as well as from healthy volunteers. Especially VEGF seems to be suitable to discriminate between non-small cell lung cancer (NSCLC) patients and control groups with highest VEGF values in EBC of patients with progressive NSCLC. The concentration of angiogenic factors correlated with disease progression as well as higher tumor stage. This study supports cytokine analysis in EBC as a suitable noninvasive diagnostic screening method for lung cancer detection and monitoring.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-Host-Disease (GvHD) induced by human immune cells. We used a model of acute GvHD by transfer of human peripheral blood mononuclear cells (PBMCs) into NSG mice. The severity of GvHD was reflected by weight loss and was associated with engraftment of human cells and the expansion of leukocytes, particularly granulocytes and monocytes. Pre-treatment of PBMCs with the anti-human CD4 antibody MAX.16H5 IgG1 or IgG4 attenuated GvHD. The transplantation of 2 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 10(7) PBMCs pre-incubated with MAX.16H5 IgG1 or IgG4, GvHD development was reduced and survival was increased. Immune reconstitution was measured by flow cytometry and confirmed for human leukocytes (CD45), CD3(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) T helper cells. Human B cells (CD19) and monocytes (CD14) could not be detected. Histopathological analysis (TUNEL assay) of the gut of recipient animals showed significantly less apoptotic crypt cells in animals receiving a MAX.16H5 IgG1 pre-incubated graft. These findings indicate that pre-incubation of an allogeneic graft with an anti-human CD4 antibody may decrease the frequency and severity of GvHD after hematopoietic stem cell transplantation (HSCT) and the need of conventional immunosuppressive drugs. Moreover, this approach most probably provides a safer HSCT that must be confirmed in appropriate clinical trials in the future. © 2016 International Society for Advancement of Cytometry.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

Acute lung injury (ALI) is characterized by increased capillary permeability, interstitial and alveolar oedema, influx of circulating inflammatory cells, and formation of hyaline membranes. Vascular endothelial growth factor (VEGF) has been correlated to a favourite prognosis in ARDS in a number of investigations. VEGF plays a role in regulating vascular permeability to water and protein. The aim of this investigation was to characterize the role of VEGF, Angiogenin, basic fibroblast growth factor (bFGF), IL-8, and TNF-alpha, lactate and pH in exhaled breath condensate (EBC) in mechanical ventilated patients with acute lung injury. For this purpose, exhaled breath condensate was collected from 30 patients with ALI at 24 to 72 hours from start of mechanical ventilation and correlated with ventilatory parameters, clinical scores, and outcome. Cytokines were measured by a cytometric bead array assay (CBA). We observed a significant lower value of VEGF in EBC in the group with lethal outcome compared to survival group (median:32.4 pg/ml vs. 108 pg/ml in survivors.; Mann-Whitney test: p<0.0001) but no significant difference for Angiogenin, bFGF, IL-8, or TNF-alpha. In addtion EBC-lactate and EBC-pH correlated with lung injury severity indices. There was a further correlation of bFGF and IL-8 in EBC with lung injury severity indices. We conclude that measurement of lactate, pH, bFGF, and VEGF in EBC may provide information on prognosis in ALI.

printing supported by . Visit Chiesi at Stand D.30 MONDAY, SEPTEMBER 26TH 2011 Results: Depending on the presence of haplotype-defining point-mutations, patients were divided into three haplotypes: H1 (n=59), H2 (n=17) and recombinant H1/H3 (n=16). ALI occurred in 53% of H2 carriers, 24% of H1 carriers and 50% of H1/H3 carriers with odds ratios 1.0, 0.284 (Confidence Interval= 0.090-0.904) and 0.963, respectively (p=0.033). Severe sepsis/septic shock was positively correlated with ALI/ARDS development, while APACHE II, SOFA, age and soluble EPCR levels were not independent predictors of ALI/ARDS. Conclusion: EPCR genotype seems to be a determinant of ALI/ARDS development in critically ill patients. H1 haplotype carriers have a reduced risk of ALI/ARDS, as compared to H2 haplotype and H1/H3 carriers. 1686 Diagnostic value of Von Willebrand factor (VWF) in patients suffering from respiratory distress Rabab El Wahsh, Safaa Amin, Enas Essa. Chest Department, Faculty of Medicine, Minoufiya University, Shebin El Kom, Minoufiya Governorate, Egypt Forensic Medicine &Clinical Toxicology Department, Faculty of Medicine, Minoufiya University, Shebin El Kom, Minoufiya Governorate, Egypt Clinical Pathology Department, Faculty of Medicine, Minoufiya University, Shebin El Kom, Minoufiya Governorate, Egypt Introduction: Acute respiratory distress syndrome (ARDS) is characterized by an extensive alveolar capillary leak. Von Willebrand Factor antigen (VWF) is a macromolecular antigen that is considered as a marker of endothelial activation. Aim: To investigate the diagnostic value of VWF antigen in patients with ALI/ARDS caused by poisoning or non-poisoning etiology. Patients and methods: VWF antigen was measured in 52 patients with ALI/ARDS, 13 poisoned patients without ALI/ARDS and 20 control subjects. Results: There was a highly significant difference between VWF level in patients and control groups (P≤ 0.001). VWF level had a significant negative correlation with the ratio between PaO2 and FIO2 in patients with respiratory distress. There was a non significant difference in VWF level between poisoned and nonpoisoned cases. There was a significant relationship between the level of VWF and the severity of poisoning in patients with respiratory distress. Among the poisoned patients, the highest level of VWF was in patients with anticholinesterase poisoning. The difference between VWF level in poisoned patients with ALI/ARDS and those without was highly significant. The level of VWF didn’t affect patients’ need for mechanical ventilation or their mortality. The cut –off value of VWF at 100% sensitivity and 20% specificity was 0.99 units, while the cut –off value of VWF at 100% specificity and 60% sensitivity was 1.87 units. Conclusion: VWF has a diagnostic value for ALI/ARDS but it does not differentiate between poisoning or non-poisoning etiology, nor does it predict outcome of the illness. VWF is significantly increased in patients with anticholinesterase poisoning. 1687 Stimulation of NOD1 induces RIP2, TAK1 and p38 MAPK dependent pro-inflammatory signalling in human lung microvascular endothelial cells Timothy L. Gatheral, Laura Moreno, Daniel M. Reed, Rekha Badiger, Lucy K. Bailey, Neil Galloway-Phillipps, Jane A. Mitchell. Cardiothoracic Pharmacology, Imperial College, London, United Kingdom Gram-negative bacteria are an important cause of septic shock. NOD1 receptors recognise peptidoglycan in the bacterial cell wall and initiate pro-inflammatory responses. We have previously shown in rodents that stimulation of NOD1 induces vascular dysfunction in vitro and profound shock in vivo. In this study we investigated the role of NOD1 in human lung microvascular endothelial cells (HMVEC) which represent a site of key importance in the pathophysiology of sepsis and acute lung injury. HMVEC from healthy donors were cultured in 96-well plates. Cells were treated for 24 hours with vehicle ± LPS (TLR4) or iE-DAP (NOD1). In additional experiments cells were pre-treated for 1 hour with specific signalling inhibitors prior to addition of agonists (n=4-5). Cell activation was assessed by multiplex ELISA and by specific ELISAs for CXCL8 and 6-keto Prostaglandin F1α. iE-DAP induced significant release of CXCL8, 6-Keto Prostaglandin F1α, IL-1β, IL-2, and IFNγ. 5Z-7-oxo-zeaenol or BIRB0796 similarly inhibited responses to iE-DAP (Figure 1) or LPS (P<0.05;two-way ANOVA). By contrast, PP2 was more potent an inhibitor of iE-DAP (Figure 1) than LPS (p<0.05; two-way ANOVA). Figure 1 In conclusion NOD1 is active in human microvascular endothelium and converges with TLR4 signalling at the level of TAK1 and p38 MAPK. NOD1 thus represents a potential target in the treatment of gram-negative sepsis. 1688 Lactate, pH and angiogenetic markers in exhaled breath condensate correlate with outcome and disease severity in patients with acute lung injury Christian Gessner1, Benedikt Malmann1, Hartmut Kuhn1, Peter Ruschpler2, Lothar Engelamnn3, Stefan Hammerschmidt1, Ulrich Sack2, Hubert Wirtz1. 1Department of Respiratory Medicine, 2Department of Immunology, 3Department of Intensive Care Medicine, University of Leipzig, Leipzig, Germany Acute lung injury (ALI) is characterized by increased capillary permeability, interstitial and alveolar oedema, influx of circulating inflammatory cells, and formation of hyaline membranes. Vascular endothelial growth factor (VEGF) has been correlated to a favourite prognosis in ARDS in a number of investigations. VEGF plays a role in regulating vascular permeability to water and protein. The aim of this investigation was to characterize the role of VEGF, Angiogenin, basic fibroblast growth factor (bFGF), IL-8, and TNF-alpha, lactate and pH in exhaled breath condensate (EBC) in mechanical ventilated patients with acute lung injury. For this purpose, exhaled breath condensate was collected from 30 patients with ALI at 24 to 72 hours from start of mechanical ventilation and correlated with ventilatory parameters, clinical scores, and outcome. Cytokines were measured by a cytometric bead array assay (CBA). We observed a significant lower value of VEGF in EBC in the group with lethal outcome compared to survival group (median:32.4 pg/ml vs. 108 pg/ml in survivors.; Mann-Whitney test: p<0.0001) but no significant difference for Angiogenin, bFGF, IL-8, or TNF-alpha. In addtion EBC-lactate and EBC-pH correlated with lung injury severity indices. There was a further correlation of bFGF and IL-8 in EBC with lung injury severity indices. We conclude that measurement of lactate, pH, bFGF, and VEGF in EBC may provide information on prognosis in ALI. 1689 Which is the best source of mesenchymal cells to treat acute lung injury? Johnatas Silva1, Ana Paz2, Debora Xisto1, Miquéias Lopes-Pacheco3, Elga Bandeira3, Milena Vasconcellos1, Vera Capelozzi4, Marcelo Morales3, Paolo Pelosi5, Elizabete Cirne-Lima2, Patricia Rocco1. 1Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Laboratory of Embryology and Cell Differentiation, Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre, Brazil; 3Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4Department of Pathology, University of Sao Paulo, Sao Paulo, Brazil; 5Dipartimento di Scienze Chirurgiche e Diagnostiche Integrate, Universita’ degli Studi di Genova, Genova, Italy Mesenchymal stem cells (MSC) may derive from bone marrow, adipose tissue or lung. Previous studies have shown that bone marrow derived mesenchymal stem cells (BM-MSC) exert beneficial effects in acute lung injury (ALI), but the effects of adipose tissue and lung derived mesenchymal cells (AD-MSC and L-MSC, respectively) have not been evaluated so far. The aim of this study was to investigate the effects of BM-MSC, AD-MSC, L-MSC on lung mechanics and morphometry, as well as inflammation and remodeling in an experimental model of ALI. Fortyeight female Wistar rats (200-250g) received Escherichia coli lipopolysaccharide (LPS) intratracheally (100 μg, ALI) or saline (C). At 48 hours, ALI and C groups were further randomized into subgroups receiving saline (0.05 mL), BM-MSC, AD-MSC, and L-MSC (1×105) intravenously. Bone marrow cells were extracted from four male Wistar rats. The induction of differentiation showed that cells from bone marrow, adipose tissue and lung were able to generate osteocytes, adipocytes and chondrocytes besides presenting CD34-, CD90+ and CD29+ profile. At day 7, mesenchymal cells promoted a reduction in lung static elastance, resistive and viscoelastic pressures, alveolar collapse, collagen fiber content and number of neutrophils in lung tissue, independent of the source. However, the beneficial effects of BM-MSC and AD-MSC on lung parenchyma remodeling were greater than those observed with L-MSC. In conclusion, in the present LPS-induced ALI model, BM-MSC and AD-MSC therapies were more effective than L-MSC at modulating inflammatory and fibrogenic processes. Supported by: CAPES, PRONEX, FAPERJ, CNPq 1690 Mesenchymal stem cells prevent early inflammation in a rat model of ventilator induced lung injury Laura Chimenti1,2 , Tomas Luque1, Marisa Bonsignore2, Daniel Navajas1,3,5, Ramon Farrè1,3,4 . 1Unit of Biophysics and Bioengineering, Faculty of Medicine, University of Barcelona, Barcelona, Spain; 2DIBIMIS, Section of Pneumology, University of Palermo, Palermo, Italy; 3CIBER de Enfermidades Respiratorias, Ciberes, Bunyola, Spain; 4Institut d’Investigacions Biomèdiques August Pi i Sunyer, IDIBAPS, Barcelona, Spain; 5Institute for Bioengineering of Catalonia, IBEC, Barcelona, Spain Background: Recent studies have suggested that bone marrow-derived mesenchy287s Oral Presentation Forum 08:30-10:30 Abstract printing supported by . Visit Chiesi at Stand D.30printing supported by . Visit Chi

Background Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4−/−, HLA-DR3+ mice. Methodology/Principal Findings Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. Conclusions/Significance NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys-X-Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys-X-Cys ligand (CXCL)9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.