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Ex-vivo lung perfusion (EVLP) has extended the number of transplantable lungs by reconditioning marginal organs. However, EVLP is performed at 37°C without homeostatic regulation leading to metabolic wastes' accumulation in the perfusate and, as a corrective measure, the costly perfusate is repeatedly replaced during the standard of care procedure. As an interesting alternative, a hemodialyzer could be placed on the EVLP circuit, which was previously shown to rebalance the perfusate composition and to maintain lung function and viability without appearing to impact the global gene expression in the lung. Here, we assessed the biological effects of a hemodialyzer during EVLP by performing biochemical and refined functional genomic analyses over a 12h procedure in a pig model. We found that dialysis stabilized electrolytic and metabolic parameters of the perfusate but enhanced the gene expression and protein accumulation of several inflammatory cytokines and promoted a genomic profile predicting higher endothelial activation already at 6h and higher immune cytokine signaling at 12h. Therefore, epuration of EVLP with a dialyzer, while correcting features of the perfusate composition and maintaining the respiratory function, promotes inflammatory responses in the tissue. This finding suggests that modifying the metabolite composition of the perfusate by dialysis during EVLP can have detrimental effects on the tissue response and that this strategy should not be transferred as such to the clinic. [ABSTRACT FROM AUTHOR]

Lung transplantation is the only curative option for end-stage chronic respiratory diseases. However the survival rate is only about 50% at 5 years. Although experimental evidences have shown that innate allo-responses impact on the clinical outcome, the knowledge of the involved mechanisms involved is limited. We established a cross-circulatory platform to monitor the early recruitment and activation of immune cells in an extracorporeal donor lung by coupling blood perfusion to cell mapping with a fluorescent marker in the pig, a commonly-used species for lung transplantation. The perfusing pig cells were easily detectable in lung cell suspensions, in broncho-alveolar lavages and in different areas of lung sections, indicating infiltration of the organ. Myeloid cells (granulocytes and monocytic cells) were the dominant recruited subsets. Between 6 and 10 h of perfusion, recruited monocytic cells presented a strong upregulation of MHC class II and CD80/86 expression, whereas alveolar macrophages and donor monocytic cells showed no significant modulation of expression. This cross-circulation model allowed us to monitor the initial encounter between perfusing cells and the lung graft, in an easy, rapid, and controllable manner, to generate robust information on innate response and test targeted therapies for improvement of lung transplantation outcome. [ABSTRACT FROM AUTHOR]

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the efficacy of S-(+)-ibuprofen on cartilage degradation as drug candidate for DDS loading. Humeral cartilage and joint capsule explants were collected from healthy sheep shoulder joints and they were cultured in mono- or in co-culture for 13 days with LPS in combination with S-(+)-ibuprofen at 50 µM and 1 mM. S-(+)-ibuprofen (50 µM) blocked prostaglandins production in LPS-activated explants but did not reduce cartilage degradation. By contrast, 1 mM S-(+)-ibuprofen treatment of cartilage explants reduced nitric oxide synthesis by 51% ( p = 0.0072), proteoglycans degradation by 35% ( p = 0.0114) and expression of serum amyloid protein - the main protein induced upon LPS challenge - by 44% ( p < 0.0001). On contrary, in presence of synovial membrane, the protective effects of S-(+)-ibuprofen on cartilage damages were significantly diminished. At 1mM, S-(+)-ibuprofen reduced the cell lysis during culture of cartilage and joint capsule either in mono- or in co-culture. This study performed on sheep explants shows that 1 mM S-(+)-ibuprofen inhibited cartilage degradation via a mechanism independent of cyclooxygenase inhibition. Reduction of prostaglandins synthesis at 50 µM in all treatment groups and reduction of cartilage degradation observed at 1 mM suggest that S-(+)-ibuprofen could be considered as a promising drug candidate for the loading of intra-articular DDS. [ABSTRACT FROM AUTHOR]

Aim: To determine whether up-regulation of basic fibroblast growth factor (bFGF) in VX2 cells reduces tumor necrosis., Materials and Methods: VX2 cells were transfected with expression vector containing cDNA of rabbit bFGF. Stable clones producing rabbit bFGF (bFGF-VX2) were selected. bFGF-VX2 (n=5) or non-transfected VX2 (control) (n=5) cells were implanted into leg muscle of 10 rabbits. The tumors were characterized 21 days after grafting., Results: Overexpression of bFGF by VX2 tumors significantly reduced necrosis (p<0.0223) and increased cell viability (p<0.0223), without effect on the mean vascular density. bFGF concentration was significantly higher in bFGF-VX2 tumors (p<0.0062) and negatively correlated with tumor volume at day 21 (ρ=-0.927, p<0.0034). Vascular endothelial growth factor concentration was significantly lower in bFGF-VX2 tumors (p<0.0105) and negatively correlated with the bFGF concentration of tumors (ρ=-0.903, p<0.0067)., Conclusion: The overexpression of bFGF in VX2 cells increased tumor viability and reduced necrosis, making the evaluation of long-term anticancer therapies possible in this model., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: To compare the cytotoxic effects of 11 anticancer agents against VX2 and HepG2 cells in order to establish candidate drugs that can be tested preclinically on VX2 tumor model for transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC)., Materials and Methods: VX2 and HepG2 cells were incubated with different drug concentrations. The half-maximal inhibitory concentration (IC50) values were determined by total cell protein assay for anthracyclines, platins, irinotecan, mytomicin-C (MMC), 5-fluorouracil (5-FU) and antiangiogenics., Results: IC50 values for VX2 and HepG2 were found close for doxorubicin (0.8 μM vs. 1.1 μM), MMC (13.9 μM vs. 8.7 μM), sunitinib (32.7 vs. 33.7 μM), sorafenib (10.3 vs. 8.9 μM), lapatinib (30 vs. 18.3 μM) and different for platins and irinotecan. Oxaliplatin was less active against VX2 than HepG2 (IC50=41 μM vs. 2.7 μM), cisplatin was more active against VX2 than HepG2 (IC50=8.0 μM vs. 15.9 μM), whereas carboplatin had a low toxicity against both cell lines (70.4 μM vs. 538.3 μM). The toxicity of 5-FU against VX2 and HepG2 was low (IC50=560.6 μM vs. 323.2 μM). Irinotecan was less active against VX2 vs. HepG2 (IC50=44.5 μM vs. 15.3 μM). Bevacizumab had no effect on either of the cell lines up to 6.7 μM., Conclusion: Drugs recommended for pre-clinical trials of TACE in the VX2 model are doxorubicin, sunitinib, sorafenib, MMC, lapatinib and 5-FU., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.