Influenza A(H5N8) Virus Similar to Strain in Korea Causing Highly Pathogenic Avian Influenza in Germany.

Highly pathogenic avian influenza (H5N8) virus, like the recently described H5N8 strain from Korea, was detected in November 2014 in farmed turkeys and in a healthy common teal (Anas crecca) in northeastern Germany. Infected wild birds possibly introduced this virus.

The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism.

RATIONALE: The neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone. OBJECTIVE: Catestatin shares several functions with angiogenic factors. We therefore reasoned that catestatin induces growth of new blood vessels. METHODS AND RESULTS: Catestatin induced migration, proliferation, and antiapoptosis in endothelial cells and exerted capillary tube formation in vitro in a Matrigel assay, and such effects were mediated via G protein, mitogen-activated protein kinase, and Akt. Catestatin-induced endothelial cell functions are further mediated by basic fibroblast growth factor, as shown by blockade of effects by a neutralizing fibroblast growth factor antibody. Furthermore, catestatin released basic fibroblast growth factor from endothelial cells and stimulated fibroblast growth factor signaling. In addition to its function on endothelial cells, catestatin also exerted effects on endothelial progenitor cells and vascular smooth muscle cells. In vivo, catestatin induced angiogenesis in the mouse cornea neovascularization assay and increased blood perfusion and number of capillaries in the hindlimb ischemia model. In addition to angiogenesis, catestatin increased density of arterioles/arteries and incorporation of endothelial progenitor cells in the hindlimb ischemia model, indicating induction of arteriogenesis and postnatal vasculogenesis. CONCLUSION: We conclude that catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism.

Gene therapy with the angiogenic cytokine secretoneurin induces therapeutic angiogenesis by a nitric oxide-dependent mechanism.

RATIONALE: The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells. OBJECTIVE: We sought to investigate the effects of secretoneurin on therapeutic angiogenesis. METHODS AND RESULTS: We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells. CONCLUSIONS: In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide-dependent mechanism.

Permeation studies on freshly excised rat gastric mucosa: influence of pH.

The objective of this study was to evaluate the influence of pH on the permeation of model drugs through freshly excised rat stomach. Additionally, the capability of excised gastric mucosa to maintain an acidic pH was assessed. In vitro permeation studies were performed in Ussing-type diffusion chambers with rat stomach using fluorescence-labeled bacitracin (bac-FITC), sodium fluorescein (NaFlu), propranolol HCl, and cimetidine as model drugs. The pH was adjusted to pH 1, 2, and 6.8 in the donor chamber and pH 7.4 in the acceptor chamber. The study demonstrated that both, the fore stomach and the glandular gastric mucosa, are capable of maintaining an acidic pH of 1-1.2 in the donor chamber. P(app) (permeation coefficients) were found to be 1.4 ± 0.6 ×·10(-7) and 7.6 ± 0.7 ×·10(-7) for bac-FITC and 3.3 ± 1.5 ×·10(-7) and 2.4 ± 0.6 ×·10(-6) cm/sec for NaFlu at pH 2 and 6.8, respectively, in the glandular stomach. In order to evaluate the effect of pH on the integrity of paracellular space, propranolol as high-permeability drug and cimetidine as low-permeability drug were chosen. The P(app) of propranolol HCl was determined to be 5.9 ± 0.3 ×·10(-7) and 1.1 ± 0.7 ×·10(-6) cm/sec at pH 2 and 6.8, respectively, in the glandular stomach. Cimetidine showed a permeability of 1.4 ± 0.4 ×·10(-5) and 9.6 ± 2.3 ×·10(-6) cm/sec at pH 2 and 6.8. Results provide essential basic information for the development of gastric drug delivery systems.

In vivo determination of the time and location of mucoadhesive drug delivery systems disintegration in the gastrointestinal tract.

OBJECTIVE: The objective of this study was to use magnetic resonance imaging (MRI) to detect the time when and the location at which orally delivered mucoadhesive drugs are released. MATERIALS AND METHODS: Drug delivery systems comprising tablets or capsules containing a mucoadhesive polymer were designed to deliver the polymer to the intestine in dry powder form. Dry Gd-DTPA [diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate] powder was added to the mucoadhesive polymer, resulting in a susceptibility artifact that allows tracking of the application forms before their disintegration and that gives a strong positive signal on disintegration. Experiments were performed with rats using T(1)-weighted spin-echo imaging on a standard 1.5-T MRI system. RESULTS: The susceptibility artifact produced by the dry Gd-DTPA powder in tablets or capsules was clearly visible within the stomach of the rats and could be followed during movement towards the intestine. Upon disintegration, a strong positive signal was unambiguously observed. The time between ingestion and observation of a positive signal was significantly different for different application forms. Quantification of the remaining mucoadhesive polymer in the intestine 3 h after observed release showed significant differences in mucoadhesive effectiveness. CONCLUSION: MRI allows detection of the exact time of release of the mucoadhesive polymer in vivo, which is a prerequisite for a reliable quantitative comparison between different application forms.

Thiolated polymers: evaluation of the influence of the amount of covalently attached L-cysteine to poly(acrylic acid).

It was the aim of this study to investigate the influence of the amount of thiol groups being covalently attached to poly(acrylic acid) 450 kDa on its properties. Five different PAA(450)-L-cysteine conjugates (PAA(450)-Cys) were synthesized bearing 53.0 (PAA I), 113.4 (PAA II), 288.8 (PAA III), 549.1 (PAA IV) and 767.0 (PAA V) micromol immobilized thiol groups per gram polymer. Mucoadhesion studies utilizing the rotating cylinder method, tensile studies and disintegration studies were performed. Self-crosslinking properties were measured by the increase in viscosity. Permeation studies were performed on rat small intestine and Caco-2 monolayers using sodium fluorescein as model drug. Following residence times on the rotating cylinder could be identified: PAA I 3.1; PAA II 5.2; PAA III 22.0; PAA IV 33.8; PAA V 53.7; control 1.3 [h]. The disintegration time of all PAA(450)-Cys tablets was strongly dependent on the degree of thiolation of the polymer. Self-crosslinking studies showed that the different PAA(450)-Cys conjugates (3% m/v) in phosphate buffer, pH 6.8, formed intramolecular disulfide bonds. In case of Caco-2 monolayer transport studies following P(app)-values could be identified: PAA I 9.8; PAA II 10.1; PAA III 11.1; PAA IV 8.9; PAA V 8.2; control 6.4 [P(app)x10(-6), cms(-1)]. Mucoadhesive and self-crosslinking properties are strongly dependent on the degree of thiolation of the polymer and with respect to transport studies, an optimum amount of covalently attached L-cysteine could be identified.

Thiomers: preparation and in vitro evaluation of a mucoadhesive nanoparticulate drug delivery system.

It was the aim of this study to develop a mucoadhesive nanoparticulate delivery system. Nanoparticles were generated by in situ gellation of the thiomer chitosan-4-thiobutylamidine (chitosan-TBA) with tripolyphosphate (TPP) followed by stabilization via the formation of inter- and intrachain disulfide bonds by oxidation with H(2)O(2) in various concentrations. Afterwards TPP was removed by exhaustive dialysis at pH 1-2. Incorporation of the model compound fluorescein diacetate (FDA) was achieved by incubation of this fluorescence marker, dissolved in acetonitrile, with aqueous particle suspensions for 1h at room temperature. Mucoadhesion studies were performed on porcine intestinal mucosa. Results showed that the preparation method described above leads to nanoparticles of a mean diameter of 268+/-15 nm and a FDA load of 2%. Due to the removal of the anionic crosslinker TPP, the zeta potential of the nanoparticles was raised from 4+/-1 up to 19+/-2 mV without loosing stability of the nanoparticles. The more H(2)O(2) was added to the particles, the more inter- and intrachain disulfide bonds were formed. The more thiol groups were oxidized within the particles, however, the lower was the improvement in mucoadhesive properties. Nevertheless, even when 91% of all thiol groups on the nanoparticles were oxidized, their mucoadhesive properties were still twice as high as the mucoadhesive properties of unmodified nanoparticles. Thiolated chitosan nanoparticles show a two-fold higher zeta potential (I), improved stability (II) and more than doubled mucoadhesive properties (III) than corresponding unmodified chitosan nanoparticles. Therefore, they seem to be advantageous over ionically crosslinked chitosan nanoparticles.

Thiomers: forms, functions and applications to nanomedicine.

Thiolated polymers or designated thiomers are gained by immobilization of sulhydryl-bearing ligands on the polymeric backbone of well established polymers, such as chitosan and poly(acrylates). This functionalization leads to significantly improved properties compared with the corresponding unmodified polymers. Mucoadhesive properties are strongly improved by the formation of disulfide bonds between thiol groups of the thiomer and cysteine-rich glycoproteins of the mucus gel layer. Moreover, enzyme- and efflux-pump inhibiting, as well as significantly improved permeation-enhancing properties, are advantages of polymer thiolization. Thiomer micro- and nano-particlulate delivery systems can be generated via different techniques, such as in situ gelation and subsequent covalent crosslinking, radical emulsion polymerization, emulsification/solvent evaporation or air jet milling. As thiomer micro- and nano-particles were shown to exhibit the same features as thiolated polymers per se, they might be useful tools for the delivery of various types of challenging drugs.