Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Dog peritoneal and pleural cavities as bioreactors to grow autologous vascular grafts

Objective: The purpose of this study was to grow artificial blood vessels for autologous transplantation as arterial interposition grafts in a large animal model (dog). Method and results: Tubing up to 250 mm long, either bare or wrapped in biodegradable polyglycolic acid (Dexon) or nonbiodegradable polypropylene (Prolene) mesh, was inserted in the peritoneal or pleural cavity of dogs, using minimally invasive techniques, and tethered at one end to the wall with a loose suture. After 3 weeks the tubes and their tissue capsules were harvested, and the inert tubing was discarded. The wall of living tissue was uniformly 1-1.5 mm thick throughout its length, and consisted of multiple layers of myofibroblasts and matrix overlaid with a single layer of mesothelium. The myofibroblasts stained for a-smooth muscle actin, vimentin, and desmin. The bursting strength of tissue tubes with no biodegradable mesh scaffolds was in excess of 2500 mm Hg, and the suture holding strength was 11.5 N, both similar to that in dog carotid and femoral arteries. Eleven tissue tubes were transplanted as interposition grafts into the femoral artery of the same dog in which they were grown, and were harvested after 3 to 6.5 months. Eight remained patent during this time. At harvest, their lumens were lined with endothelium-like cells, and wall cells stained for alpha-actin, smooth muscle myosin, desmin and smoothelin; there was also a thick adventitia containing vasa vasorum. Conclusion: Peritoneal and pleural cavities of large animals can function as bioreactors to grow myofibroblast tubes for use as autologous vascular grafts.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

Bioresorbable vascular scaffold treatment induces the formation of neointimal cap that seals the underlying plaque without compromising the luminal dimensions: a concept based on serial optical coherence tomography data.

Aims: To evaluate the implications of an Absorb bioresorbable vascular scaffold (Absorb BVS) on the morphology of the superficial plaques. Methods and results: Forty-six patients who underwent Absorb BVS implantation and 20 patients implanted with bare metal stents (BMS) who had serial optical coherence tomographic examination at baseline and follow-up were included in this analysis. The thin-capped fibroatheromas (TCFA) were identified in the device implantation regions and in the adjacent native coronary segments. Within all regions, circumferential locations of TCFA and calcific tissues were identified, and the neointimal thickness was measured at follow-up. At six to 12-month follow-up, only 8% of the TCFA detected at baseline were still present in the Absorb BVS and 27% in the BMS implantation segment (p=0.231). Sixty percent of the TCFA in native segments did not change their phenotype at follow-up. At short-term follow-up, significant reduction in the lumen area of the BMS was noted, which was higher compared to that reported in the Absorb BVS group (-2.11±1.97 mm2 vs. -1.34±0.99 mm2, p=0.026). In Absorb BVS, neointima tissue continued to develop at midterm follow-up (2.17±0.48 mm2 vs. 1.38±0.52 mm2, p<0.0001) and covered the underlying tissues without compromising the luminal dimensions (5.93±1.49 mm2 vs. 6.14±1.49 mm2, p=0.571) as it was accommodated by the expanded scaffold (8.28±1.74 mm2 vs. 7.67±1.28 mm2, p<0.0001). Conclusions: Neointimal tissue develops following either Absorb BVS or BMS implantation and shields lipid tissues. The neointimal response in the BMS causes a higher reduction of luminal dimensions compared to the Absorb BVS. Thus, Absorb BVS may have a value in the invasive re-capping of high-risk plaques.

Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42 +/- 3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5 +/- 3.5%, of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Polymer stent coating for prevention of neointimal hyperplasia

AIMS: Restenosis has been the principal limitation of bare metal stents. Based upon the presumption that platelet and inflammatory cell recruitment initiate neointimal proliferation, we explored a novel polymer coating that reduces cell-stent interactions. The purpose of the present study was to investigate the effect of poly(L-lysine)-graft-poly(ethyleneglycol) (PLL-g-PEG) adsorbed to stent surfaces to reduce neointimal hyperplasia in the porcine restenosis model. METHODS AND RESULTS: Seven animals were instrumented each with 2 stainless steel stents (15 mm length, 2.5-3.5 mm diameter), randomly implanted in 1 major epicardial coronary artery. One stent was dip-coated with PLL-g-PEG, whereas the other stent served as the uncoated control stent. All animals were sacrificed after 6 weeks for histological examination. Neointimal hyperplasia was significantly less (-51%) in the PLL-g-PEG-coated stents (1.15 +/- 0.59 mm2) than in the uncoated control stents (2.33 +/- 1.01 mm2; p < 0.001). Conversely, lumen size was larger in the PLL-g-PEG-coated stents (2.91 +/- 1.17 mm2) than in the uncoated stents (2.04 +/- 0.64 mm2; p < 0.001). High magnification histomorphologic examination revealed no signs of inflammation or thrombus formation in either stent group. CONCLUSIONS: Polymeric steric stabilization of stents with PLL-g-PEG significantly reduces neointimal hyperplasia in the porcine restenosis model. Reduction of cell-stent interactions mediated by PLL-g-PEG appear to improve biocompatibility of stainless steel stents without evidence of adverse inflammatory or prothrombotic effects.

Role of endogenous Fas (CD95/Apo-1) ligand in balloon-induced apoptosis, inflammation, and neointima formation

BACKGROUND: Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. METHODS AND RESULTS: To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). CONCLUSIONS: Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

Differential effects of chlorinated and oxidized phospholipids in vascular tissue:implications for neointima formation

The presence of inflammatory cells and MPO (myeloperoxidase) in the arterial wall after vascular injury could increase neointima formation by modification of phospholipids. The present study investigates how these phospholipids, in particular oxidized and chlorinated species, are altered within injured vessels and how they affect VSMC (vascular smooth muscle cell) remodelling processes. Vascular injury was induced in C57BL/6 mice and high fat-fed ApoE-/- (apolipoprotein E) mice by wire denudation and ligation of the left carotid artery (LCA). Neointimal and medial composition was assessed using immunohistochemistry and ESI-MS. Primary rabbit aortic SMCs (smooth muscle cells) were utilized to examine the effects of modified lipids on VSMC proliferation, viability and migration at a cellular level. Neointimal area, measured as intima-to-media ratio, was significantly larger in wire-injured ApoE-/- mice (3.62±0.49 compared with 0.83±0.25 in C57BL/6 mice, n=3) and there was increased oxidized low-density lipoprotein (oxLDL) infiltration and elevated plasma MPO levels. Relative increases in lysophosphatidylcholines and unsaturated phosphatidylcholines (PCs) were also observed in wire-injured ApoE-/- carotid arteries. Chlorinated lipids had no effect on VSMC proliferation, viability or migration whereas chronic incubation with oxidized phospholipids stimulated proliferation in the presence of fetal calf serum [154.8±14.2% of viable cells at 1 μM PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) compared with control, n=6]. In conclusion, ApoE-/- mice with an inflammatory phenotype develop more neointima in wire-injured arteries and accumulation of oxidized lipids in the vessel wall may propagate this effect.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Culturable Bacterial Diversity From A Feed Water Of A Reverse Osmosis System, Evaluation Of Biofilm Formation And Biocontrol Using Phages.

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

Epiretinal Membrane Formation Associated With Idiopathic Macular Telangiectasia: Case Report.

A 46-year-old woman complained of blurred and distorted vision in both eyes. Ophthalmic examination showed that visual acuity was 20/200 for the right eye and counting fingers left eye. Fundoscopy revealed perimacular hemorrhages, aneurismal dilatation of the vessels in the posterior pole, and a white and elevated lesion adjacent to vascular changes. We report a case of idiopathic macular telangiectasia and epiretinal membrane that occurs concomitantly. To our knowledge, this is the first report that describes an association between idiopathic macular telangiectasia and epiretinal membrane formation.