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Biofilm elimination is often necessary during antimicrobial therapy or industrial medical manufacturing decontamination. In this context, ultrasound treatment has been frequently described in the literature for its antibiofilm effectiveness, but at the same time, various authors have described ultrasound as a formidable enhancer of bacterial viability. This discrepancy has found no solution in the current literature for around 9 years; some works have shown that every time bacteria are exposed to an ultrasonic field, both destruction and stimulation phenomena co-exist. This co-existence proves to have different final effects based on various factors such as: ultrasound frequency and intensity, the bacterial species involved, the material used for ultrasound diffusion, the presence of cavitation effects and the forms of bacterial planktonic or biofilm. The aim of this work is to analyze current concepts regarding ultrasound effect on prokaryotic cells, and in particular ultrasound activity on bacterial biofilm., (Copyright © 2013. Published by Elsevier B.V.)

Previous studies have shown that a recombinant vaccine expressing four highly conserved influenza virus epitopes has a potential for a broad spectrum, cross-reactive vaccine; it induced protection against H1, H2 and H3 influenza strains. Here, we report on the evaluation of an epitope-based vaccine in which six conserved epitopes, common to many influenza virus strains are expressed within a recombinant flagellin that serves as both a carrier and adjuvant. In an HLA-A2.1 transgenic mice model, this vaccine induced both humoral and cellular responses and conferred some protection against lethal challenge with the highly pathogenic H5N1 avian influenza strain. Hence, it is expected to protect against future strains as well. The data presented, demonstrate the feasibility of using an array of peptides for vaccination, which might pave the way to an advantageous universal influenza virus vaccine that does not require frequent updates and/or annual immunizations.

We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.

We have studied the Ca(2+) dynamics of bursting-spiking neurons in the lobster stomatogastric ganglion. Neurons in this ganglion undergo spontaneous oscillations in membrane voltage with a period of 1-10 s in situ. We found that neurons isolated from the ganglion and filled with the fluorescent calcium indicator Fluo-4 show simultaneous changes of membrane potential and cytoplasmic Ca(2+) concentration ([Ca(2+)](I)). These Ca(2+) signals are highly heterogeneous both in terms of amplitude and time constants. They showed variable spatial distributions with the soma exhibiting low and slow signals, and a region in the process with large and fast signals. Ca(2+) transients in the processes are dependent on external Ca(2+) and can be blocked by Co(2+), but not other, more specific Ca(2+) current blockers. Rather, nifedipine a known Ca(2+) current blocker, affects the distribution of the Ca(2+) signal, which suggests a specific localization of Ca(2+) channels. Although the signal is not absolutely dependent on action potentials, it is greatly reduced when action potentials are blocked by tetrodotoxin. Termination of the signal depends only slightly on Ca(2+) buffering mechanisms such as mitochondria, Ca(2+)/Na(+) and Ca(2+)/H(+) exchangers. We also demonstrate the presence of caffeine-sensitive internal stores in stomatogastric ganglion cells. The store distribution is different but overlaps with the voltage-dependent distribution. The maximal caffeine-activated Ca(2+) signal is in the soma and it is smaller in the processes. Unlike the voltage-activated Ca(2+) signal this signal is not blocked by Co(2+). Nevertheless, the two types of signal interact during caffeine application. This unique spatial separation of two Ca(2+) sources may have important functional implication.

We had reported that in the ischemic heart, locally formed bradykinin (BK) and angiotensin II (Ang II) activate B2- and AT1-receptors on sympathetic nerve terminals (SNE), promoting reversal of the norepinephrine (NE) transporter in an outward direction (i.e., carrier-mediated NE release). Although both BK and Ang II contribute to ischemic NE release, Ang II is likely to play a more important role. Since BK is formed by ischemic SNE, we questioned whether cardiac SNE also contribute to local Ang II formation, in addition to being a target of Ang II. SNE were isolated from surgical specimens of human right atrium and incubated in ischemic conditions. These SNE released large amounts of endogenous NE via a carrier-mediated mechanism, as evidenced by the inhibitory effect of desipramine on this process. Moreover, two renin inhibitors, pepstatin-A and BILA 2157 BS, the ACE inhibitor enalaprilat and the AT1-receptor antagonist EXP3174 prevented ischemic NE release. Western blot analysis revealed the presence of renin in cardiac SNE. Renin abundance increased more than three-fold during ischemia. Thus, renin is present in cardiac SNE and is activated during ischemia, eventually culminating in Ang II formation, stimulation of AT1-receptors and carrier-mediated NE release. Our findings uncover a novel autocrine mechanism, by which Ang II, formed at SNE in myocardial ischemia, elicits carrier-mediated NE release by activating prejuntional AT1-receptors.

Recently, we discovered a series of peripheral acting selective hypotensive vasopressin peptides. Whether these peptides may interact with receptors outside the vasopressin receptor family and affect cardiac function could not be excluded. Accordingly, we tested the effects of these hypotensive vasopressin peptides on blood pressure and heart rate in intact rats and on the heart rate, ventricular contractile force and coronary flow of isolated perfused rat hearts. We found that the hypotensive vasopressin peptides did not modify cardiac function, either in vivo or in vitro. The vasodepressor potency was reduced when assayed in rats with vasopressin-maintained baseline blood pressure, suggesting that vasopressin and the hypotensive peptide compete for a common vasodilating vasopressin receptor in the vasculature. We have now synthesized more potent and radioiodinatable hypotensive peptides that could serve as lead compounds for the development of a radiomarker for the putative vasodilating vasopressin receptor.

The serotonergic metacerebral giant cell (C1) of Helix pomatia was isolated with its bifurcate axon and plated in culture under five conditions: (i) with no target; (ii) with the appropriate target B2 near the stump of the bigger branch (CBC); (iii) with B2 near the stump of the smaller branch (CC); (iv) with a wrong target (C3) near the stump of the CBC branch and (v) with B2 and C3 positioned near the CBC and CC stump, respectively. The counting of anti-serotonin antibody-labelled varicosities of the C1 neuron showed that the presence of the appropriate target in either axonal domain both down-regulated the number of varicosities of the contralateral neuritic field, and increased their average size, whereas the wrong target induced an overall reduction of the number of C1 neuron varicosities, and inhibited the evoked transmitter release. The action potential-evoked calcium concentration increase in the neuritic terminals of the C1 neuron cultured alone, or in presence of the appropriate target, reached a value significantly higher than that reached in presence of the wrong target. These results provide evidence that the postsynaptic neuron regulates both morphological and functional development of presynaptic terminals.

GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) and GT-2331 ((1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole) were developed as new potent histamine H3 receptor antagonists. The functional activity of these ligands on the histamine H3 receptor-mediated inhibition of neurogenic contraction of the guinea-pig jejunum and histamine H3 receptor-mediated inhibition of norepinephrine release from guinea-pig heart synaptosomes were investigated. GT-2227 and GT-2331 both antagonized the inhibitory effects of (R)-alpha-methylhistamine on the contraction induced by electrical field stimulation in the guinea-pig jejunum with pA2 values of 7.9+/-0.1 and 8.5+/-0.03, respectively. In addition, GT-2227 and GT-2331 antagonized the inhibition of norepinephrine release in cardiac synaptosomes by GT-2203 ((1R,2R)-trans-2-(1H-imidazol-4-yl)cyclopropylamine), a histamine H3 receptor agonist. The current results demonstrate the antagonist activity for both GT-2227 and GT-2331 in two functional assays for histamine H3 receptors.

Synthetic vaccines utilize specific antigenic epitopes in order to elicit a protective immune response. In this work we examined the immunogenicity of chimeric proteins expressing influenza epitopes and their ability, as single products or in various combinations, to protect mice from viral challenge. Oligonucleotides coding for three epitopes (HA91-108, NP55-69 and NP147-158) stimulating B cells, T helper cells and cytotoxic T lymphocytes (CTLs), respectively, were individually inserted into the flagellin gene of a Salmonella vaccine strain. Immunization of mice with the resultant hybrid flagella resulted in a specific humoral or cellular response. The protective efficacy of the chimeric flagella was evaluated by intranasal immunization of mice, without any adjuvant, and subsequent challenge with infectious virus. The construct containing the B-cell epitope by itself led to partial protection. However, the addition of the two T-cell epitopes augmented the protection in a significant manner. The protective immunity conferred by this combined vaccine, comprising the three epitopes, persisted for at least 7 months after the last boost, and was effective against several influenza A strains. Furthermore, this vaccine fully protected mice from a lethal challenge, and enhanced their recovery process. Our results indicate that stimulation of the different arms of the immune system is required for effective anti-influenza response, and demonstrate the applicability of such synthetic recombinant approach for preparing a broad spectrum influenza vaccine.

Synthetic vaccines that are based on peptides representing immunogenic epitopes require a carrier molecule as well as an adjuvant in order to be effective. The choice of carriers or adjuvants approved for use in humans is very limited, and a considerable effort is devoted to develop new and efficient delivery systems. One of these vehicles utilizes preparations of outer membranes of meningococci, that form hydrophobic interactions, denoted proteosomes. Immunogenic proteins and peptides can be anchored to these proteosomes vesicles, which may serve as both carrier and adjuvant functions. In the present study we examined the ability of proteosomes to present epitopes of influenza, to elicit specific anti-influenza responses and to protect mice against viral challenge after intranasal immunization. Three influenza peptides were used--one corresponding to amino acid residues 91-108 of the haemagglutinin surface glycoprotein of H3 subtype, which comprises a B-cell epitope, and two from the internal nucleoprotein--a T-helper cell (Th) epitope (residues 55-69) and a cytotoxic T-lymphocyte (CTL) epitope (147-158). Mice were immunized intranasally (i.n.) with preparations containing each of the above epitopes, or various combinations thereof. The results obtained with this system demonstrate that influenza epitopes represented by synthetic peptides anchored to a proteosome carrier elicit both humoral and cellular specific immune responses, that can lead to partial protection of the mice from viral challenge. The importance of immunizing with vaccines containing both B- and T-cell peptide epitopes was emphasized by the demonstration that such vaccines elicited longer lasting immunity and led to more effective protection against influenza viral challenge.

Myocardial hypoxia is known to be accompanied by the release of atrial natriuretic factor (ANF), a peptide which dilates the coronary vessels by stimulating particulate guanylyl cyclase. We have assessed whether ANF plays a paracrine role in hypoxic coronary vasodilatation, a reaction which we had previously found to be associated with increased cyclic GMP production. Compound HS 142-1 (100 micrograms/ml), a specific antagonist of the guanylyl cyclase ANF receptor, inhibited by 50-70% the coronary-vasodilating effects of human ANF (1-10 micrograms) administered to isolated guinea pig hearts, but affected neither hypoxic coronary vasodilation nor cyclic GMP overflow. In contrast, the nitric oxide synthase inhibitor N omega-methyl-L-arginine (300 microM) reduced hypoxic coronary vasodilatation and cyclic GMP overproduction by approximately 70% and 50-60%, respectively. Thus, unlike nitric oxide, ANF appears not to play a paracrine role in hypoxic coronary vasodilatation.

The influenza virus haemagglutinin epitope 91-108, which is a conserved amino acid sequence in all type A H3 strains, was expressed in Salmonella flagellin, to evaluate its potential as a vaccine. For that purpose, a synthetic oligonucleotide comprising 54 bases coding for the corresponding sequence was inserted into the plasmid pLS408 and transformed into Escherichia coli JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative 'live vaccine' aroA mutant of Salmonella dublin. Rabbits immunized either with the live recombinant S. dublin or with the flagellin isolated from it, showed significant levels of IgG response against the synthetic peptide 91-108 as well as against the intact A/Texas/77 influenza virus. Mice immunized with the same preparations developed influenza-specific IgG antibodies in the blood and secreted IgA antibodies in their lungs. Furthermore, these mice showed about 50% protection against challenge infection with the virus. The most successful results were achieved by intranasal immunization with the isolated recombinant flagellin, when employed without the aid of adjuvant.

The purpose of our investigation was to assess the role of the endothelium in the vasomotor effects of leukotrienes. Norepinephrine-preconstricted rings isolated from guinea pig main pulmonary artery and thoracic aorta responded to LTC4 and LTD4 with a concentration-dependent relaxation. In endothelium-denuded rings, both LTC4 and LTD4 caused a concentration-dependent contraction. The LTD4 receptor antagonist ICI 198,615 inhibited both LTC4- and LTD4-induced relaxation and contraction. Inhibition of gamma-glutamyl transpeptidase with AT-125 prevented the effects of LTC4, but not those of LTD4. The relaxant effect of LTD4 was not modified by indomethacin, but was abolished by methylene blue. We conclude that: 1) LTD4 induces a receptor-mediated endothelium-dependent relaxation of cavian pulmonary artery and aorta; 2) the vasorelaxant effect of LTC4 requires its conversion to LTD4; 3) the vasorelaxant effect of LTD4 is unrelated to PGI2 release, and is probably due to the release of an "EDRF"; 4) the removal of the endothelium reveals a direct receptor-mediated vasoconstricting effect of leukotrienes.

The effect of metiamide on the cardiotoxicity produced by ouabain was studied in pentobarbital-anesthetized cats. The onset of ouabain-induced ventricular tachycardia and fibrillation was significantly delayed in cats treated with metiamide as compared with cats that did not receive metiamide. Although the mechanism by which metiamide inhibits ouabain toxicity is speculative, the data suggest that histamine H2-receptor blocking agents may be useful as anti-arrhythmic drugs in digitalis cardiotoxicity.

In the isolated guinea pig heart, histamine H1-receptor antagonists inhibit the histamine-induced negative dromotropic effect, but not the positive inotropic and chronotropic effects (Levi and Kuye, 1974, European J. Pharmacol. 27, 330). Burimamide, the H2-receptor antagonist, inhibits the positive chronotropic effect of histamine (Black et al., 1972, Nature 236, 385). In this study, the hypothesis that H1- and H2-receptors selectively mediate the various cardiac effects of histamine was tested: (a) by investigating the inhibition of cardiac histamine effects by burimamide; (b) by studying the cardiac effects of 2-methylhistamine (an H1-agonist) and 4-methylhistamine (an H2-agonist). Burimamide, as a function of concentration, inhibited the positive inotropic and chronotropic effects of histamine, whereas it attenuated the negative dromotropic effect of histamine at the higher concentration only. 4-Methylhistamine caused dose-dependent positive inotropic and chronotropic but not negative dromotropic effects; conversely, with 2-methylhistamine the negative dromotropic effect prevailed. Sinus tachycardia and slowing of atrioventricular conduction were also obtained in the guinea pig in vivo by the i.v. administration of histamine. Burimamide selectively antagonized the positive chronotropic effect, whereas promethazine (an H1-antagonist) selectively inhibited the negative dromotropic effect. These results substantiate the hypothesis that H2-receptors mediate the histamine-induced increase in rate and contractility, whereas H1-receptors mediate the slowing of atrioventricular conduction.

To further characterize the receptor mediating histamine-induced impairment of atrioventricular conduction, the effects of two selective histamine H1-receptor agonists, 2-(2-pyridyl)-ethylamine (PEA) and 2-(2-thiazolyl)-ethylamine (ThEA), were investigated using the isolated guinea pig heart. These effects were compared with those of histamine and other selective agonists. PEA and ThEA produced a weak stimulation of cardiac rate and contractility; however, they produced a marked prolongation of atrioventricular conduction. The orders of relative potencies observed substantiate the hypothesis that H2-receptors mediate the positive inotropic and chronotropic effects and H1-receptors mediate the negative dromotropic effect of histamine.

Immunologic immediate hypersensitivity reactions of the heart are characterized by tachycardia and arrhythmias caused by the release of endogenous cardiac histamine. The contribution of SRS-A to anaphylactic cardiac dysfunction was indirectly assessed by the use of the selective SRS-A receptor antagonist FPL 55712. In the presence of FPL 55712 the concentration-response curves for the chronotropic and arrhythmogenic effects of endogenous cardiac histamine were progressively shifted to the right as a function of the concentration of FPL 55712. Moreover, the duration of the positive chronotropic effect of endogenous cardiac histamine was shortened by FPL 55712. On the other hand, FPL 55712 failed to reduce the chronotropic and arrhythmogenic effects of exogenous histamine. Partially purified histamine-free SRS-A opbtained from rat peritoneum potentiated and greatly prolonged the positive chronotropic effect of exogenous histamine. Potentiation and prolongation were both blocked by FPL 55712. Thus, our results suggest that SRS-A sensitized the heart to the tachyarrhythmic effects of histamine.