Specific egg yolk antibodies (IgY) confer protection against Acinetobacter baumannii in a murine pneumonia model.

AIM: Acinetobacter baumannii, an increasingly serious health threat, is considered as one of the six most dangerous microbes of high mortality rate. However, treatment of its infections is difficult because of the lack of efficient antibiotic or commercial vaccines. Passive immunization through administration of specific antibodies such as IgY, could be an attractive practical solution. METHODS AND RESULTS: In the current study, antigenicity of two recombinant outer membrane proteins (OmpA and Omp34) as well as inactivated whole cell of A. baumannii was assessed by ELISA. Moreover, prophylactic effects of specific IgY antibodies (avian antibody) raised against these antigens were evaluated in a murine pneumonia model. The specific IgY antibodies had various prophylactic effects in the pneumonia model. OmpA was the most potent antigen in terms of triggering antibody and conferring protection. While a synergic effect was observed in ELISA for antibodies raised against a combination of OmpA and Omp34 (which are known as Omp33-36 and Omp34 kDa), an antagonistic effect was unexpectedly seen in challenges. The reason for this phenomenon remains to be precisely addressed. CONCLUSION: All the specific IgY antibodies could protect mice against pneumonia caused by A. baumannii. SIGNIFICANCE AND IMPACT OF THE STUDY: The specific IgY antibodies could be employed as a pharmaceutical against pneumonia caused by A. baumannii.

Macrophage migration inhibitory factor downregulation: a novel mechanism of resistance to anti-angiogenic therapy.

Anti-angiogenic therapies for cancer such as VEGF neutralizing antibody bevacizumab have limited durability. While mechanisms of resistance remain undefined, it is likely that acquired resistance to anti-angiogenic therapy will involve alterations of the tumor microenvironment. We confirmed increased tumor-associated macrophages in bevacizumab-resistant glioblastoma patient specimens and two novel glioblastoma xenograft models of bevacizumab resistance. Microarray analysis suggested downregulated macrophage migration inhibitory factor (MIF) to be the most pertinent mediator of increased macrophages. Bevacizumab-resistant patient glioblastomas and both novel xenograft models of resistance had less MIF than bevacizumab-naive tumors, and harbored more M2/protumoral macrophages that specifically localized to the tumor edge. Xenografts expressing MIF-shRNA grew more rapidly with greater angiogenesis and had macrophages localizing to the tumor edge which were more prevalent and proliferative, and displayed M2 polarization, whereas bevacizumab-resistant xenografts transduced to upregulate MIF exhibited the opposite changes. Bone marrow-derived macrophage were polarized to an M2 phenotype in the presence of condition-media derived from bevacizumab-resistant xenograft-derived cells, while recombinant MIF drove M1 polarization. Media from macrophages exposed to bevacizumab-resistant tumor cell conditioned media increased glioma cell proliferation compared with media from macrophages exposed to bevacizumab-responsive tumor cell media, suggesting that macrophage polarization in bevacizumab-resistant xenografts is the source of their aggressive biology and results from a secreted factor. Two mechanisms of bevacizumab-induced MIF reduction were identified: (1) bevacizumab bound MIF and blocked MIF-induced M1 polarization of macrophages; and (2) VEGF increased glioma MIF production in a VEGFR2-dependent manner, suggesting that bevacizumab-induced VEGF depletion would downregulate MIF. Site-directed biopsies revealed enriched MIF and VEGF at the enhancing edge in bevacizumab-naive patients. This MIF enrichment was lost in bevacizumab-resistant glioblastomas, driving a tumor edge M1-to-M2 transition. Thus, bevacizumab resistance is driven by reduced MIF at the tumor edge causing proliferative expansion of M2 macrophages, which in turn promotes tumor growth.

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I.

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.

Dietary fish oil prevents asynchronous contractility and alters Ca(2+) handling in adult rat cardiomyocytes.

This study examined the effects of dietary incorporation of n-3 polyunsaturated fatty acids (PUFAs) into cardiac membrane phospholipids on Ca(2+) handling (using Fura-2) and arrhythmic contractility in electrically-stimulated, adult rat ventricular cardiomyocytes. Dietary lipid supplementation with fish oil (FO) for 3 weeks significantly increased the proportion of total n-3 polyunsaturated fatty acids (in particular, docosahexaenoic acid) in ventricular membrane phospholipids compared with a saturated fat (SF) supplemented diet (26.2 +/- 0.9% vs 6.9 +/- 0.9%, respectively, P < 0.001). Cardiomyocytes isolated from the FO group were significantly (P < 0.001) less susceptible to isoproterenol-induced arrhythmic contractile activity compared with the SF group over a range of isoproterenol concentrations. Isoproterenol (0.5 &mgr;M) stimulation increased end-diastolic and systolic [Ca(2+)](i) to a similar extent in both groups. The time constant of Ca(2+) transient decay was significantly increased in the FO group compared with the SF group (98.4 +/- 2.8 ms, n = 8 and 86.9 +/- 2.1 ms, n = 8, P < 0.01, respectively). The effect of dietary n-3 PUFA incorporation into membrane phospholipids was not associated with changes in sarcoplasmic reticulum Ca(2+) content (measured by rapid application of caffeine) or membrane fluidity. The increase in the time constant of decay of Ca(2+) transients following dietary supplementation with FO may indicate altered functioning of the sarcolemmal Na(+)-Ca(2+) exchanger by n-3 PUFA incorporation into membrane phospholipids.

Effects of dietary n-3 fatty acids on contractility, Na+ and K+ currents in a rat cardiomyocyte model of arrhythmia.

The n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent ventricular fibrillation in human clinical studies and in studies involving experimental animals and isolated cardiomyocytes. This study aimed to determine whether dietary n-3 PUFAs could prevent isoproterenol and free radical-induced arrhythmic (asynchronous) contractile activity in adult rat cardiomyocytes and whether whole-cell Na(+) and K(+) currents measured by patch-clamp techniques were affected. Dietary supplementation with fish oil for 3 weeks significantly increased the proportion of total n-3 PUFAs in ventricular membrane phospholipids compared with saturated fat supplementation (18.8 +/- 0.6% vs. 8.1 +/- 1.0%, respectively). Cardiomyocytes from the fish oil group were less susceptible to isoproterenol-induced asynchronous contractile activity than were those from the saturated fat group [EC(50) values: 892 +/- 130 nM, n = 6 and 347 +/- 91 nM, n = 6 (P < 0.05), respectively]. Fish oil supplementation also prolonged the time taken to develop asynchronous contractile activity induced by superoxide and hydrogen peroxide. The voltage dependence of inactivation of Na(+) currents were significantly altered (-73.5 +/- 1.2 mV, n = 5 vs. -76.7 +/- 0.7 mV, n = 5, P < 0.05, for saturated fat and fish oil treated groups, respectively). The voltage dependence of activation of Na(+) and K(+) currents was not significantly affected by the dietary fish oil treatment. These results demonstrate the antiarrhythmic effects of dietary fish oil in a cardiomyocyte model of arrhythmia.

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids.

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 microM isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 microM, compared with no fatty acid addition (from 99.0+/-1.0% to 30.7+/-5.2% (p < 0.05) for DHA and 23.8+/-2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7+/-1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r(ss)) of atrial myocytes compared with no addition of fatty acid (control) (from r(ss) = 0.203+/-0.004 to 0.159+/-0.004 (p < 0.01) for DHA, 0.166+/-0.001 (p < 0.01) for EPA and 0.186+/-0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.

Membrane fluidity changes are associated with the antiarrhythmic effects of docosahexaenoic acid in adult rat cardiomyocytes.

Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a beta-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 +/- 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 +/- 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 +/- 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 +/- 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r(ss)) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r(ss); however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.

Antiarrhythmic fatty acids and antioxidants in animal and cell studies.

From the animal and cellular studies that will be discussed in this review, it is apparent that dietary fatty acids and antioxidants play an important role in influencing the development of ventricular tachycardia and potentially lethal ventricular fibrillation. It is this latter disturbance to the rhythmic beating of the heart that is responsible for much of the mortality from coronary heart disease. It is now recognized that diets high in certain polyunsaturated fatty acids (PUFAs) and diets containing antioxidants can afford considerable protection to the heart with regard to the generation of disorders of contractile rhythmicity. The mechanism by which such dietary components confer their cardioprotective effects are now being intensively investigated, particularly with respect to their possible effects on the molecular mechanisms underlying the excitation-contraction coupling process of the myocardial cell. This overview will cover recent studies that have focused on the antiarrhythmic role of PUFAs, particularly those of the n-3 (or omega 3) class with emphasis on experiments performed using laboratory animals, isolated heart preparations, and isolated heart cells (cardiomyocytes). The role of free radicals (reactive oxygen species) and antioxidants in disorders of cardiac rhythm also will be addressed within the perspective of reperfusion injury to the myocardium following ischemia. Emphasis will be placed on the cardioprotective role of nutritional factors and components and the possible cellular mechanisms by which such components may act.