Influence of a Probiotic Strain of Enterococcus faecium on Salmonella enterica Serovar Typhimurium DT104 Infection in a Porcine Animal Infection Model

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.The problem of increasing microbial resistance to antibiotics resulting from years of overuse and the resulting ban on the use of antibiotics in animal production have led to increased interest in alternatives to antibiotics in animal production. In recent years, probiotic bacteria have been considered as an alternative means of reducing pathogen loads in animal breeding and production units. However, while a number of studies have focused on the mode of action of probiotics, the mode of action these bacteria is not fully understood yet.A recent interdisciplinary research study of the modes of action of probiotics in swine showed that Enterococcus faecium NCIMB 10415 reduced the pathogenic bacterial load of healthy piglets (20, 26, 30, 36). In vitro studies further demonstrated that this E. faecium probiotic strain decreased the rate of invasion of a porcine intestinal epithelial cell line by Salmonella enterica serovar Typhimurium. To determine whether probiotics also provide a measure of protection during infections, experimental challenge studies with pathogenic bacteria at a defined infectious dose and under comparable conditions seem to be necessary. Field studies could be more representative of the real situation; however, the infection pressure is too low and difficult to define, and systematic sampling cannot be done.Studies of larger domestic and production animals are rare. Most such studies deal with the mode of action of probiotics in the healthy host, and only a few studies have investigated the mode of action in the context of infections with pathogenic bacteria, such as Salmonella.In a related study, weaned piglets were fed a mixture of five probiotic strains (one Pediococcus strain and four Lactobacillus strains) and challenged with Salmonella serovar Typhimurium (7). In that study, reduced incidence, severity, and duration of diarrhea and a reduced microbiological load of Salmonella were observed. Fedorka-Cray et al. (11) observed reduced numbers of Salmonella bacteria in cecal contents and at the ileocolic junction in S. enterica serovar Choleraesuis-challenged weaning piglets fed a competitive exclusion culture. In vitro investigations showed that Enterococcus strains have inhibitory effects on the growth of S. enterica serovar Enteritidis, and these effects were explained by both enterotoxin and nonenterotoxin factors (37). Other studies showed that E. faecium may be beneficial to the adhesion and colonization of Clostridium jejuni in the canine intestine (29) and reduced the rate of carryover infections with obligate intracellular pathogens from infected sows in piglets (26). E. faecium has also been shown to influence the composition of the bacterial community in the avian, swine, and canine gastrointestinal tracts (25, 29, 36).Infections with S. enterica are some of the most important sources of human gastroenteritis (39). In Germany, 52,563 human salmonellosis cases were reported in 2006 (http://www3.rki.de/SurvStat). The consumption of contaminated pork and pork products was found to be associated with 20% of human salmonellosis cases in Germany (33), indicating the importance of meat or meat products as a potential source of infection for consumers. Salmonella serovar Typhimurium, especially phage type DT104, is the Salmonella serotype most frequently isolated from pork (27), and it is of particular concern because of its acquisition of multiple antibiotic resistance (1, 38).In this study, we investigated the effect of E. faecium NCIMB 10415 on the infection dynamics of Salmonella serovar Typhimurium DT104, fecal shedding, and the patterns of Salmonella distribution in internal organs, as well as on the humoral immune response to Salmonella in weaning piglets. To the best of our knowledge, this is the first experimental study of the mode of action of a probiotic strain of E. faecium in which dissemination to different internal organs was investigated using weaned piglets experimentally infected with Salmonella.     

Prominent collagen type VI expression in juvenile angiofibromas

The extracellular matrix component collagen type VI demonstrates potent growth-stimulatory effects and has been associated with aggressive tumour growth. Although, juvenile angiofibromas (JAs) often exhibit an aggressive growth pattern, the collagen type VI expression of this fibrovascular tumour has not been addressed so far. RT-PCR, Western blot analysis and immunohistochemistry were used in this study to analyse collagen type VI, type VI collagen receptor subunits (integrin α1, α2, α10, α11 and β1) and the type VI collagen receptor NG2 in JAs (N = 15) and nasal mucosa (NM, N = 8) samples. The mRNA expression of all three collagen type VI chains was found to be up-regulated significantly (P < 10⁻³–10⁻⁵, adjusted) in JAs compared to NM tissues. The Western blot analysis proved highly prominent collagen-type VI expression in JAs. The ApoTome technique revealed strong collagen-type VI signals in tumour endothelium. NG2 (P < 10⁻³, adjusted) and α11-integrin (P = 0.04, adjusted) showed a significantly higher mRNA expression levels in JAs than in NM samples. NG2, α1-, α2- and β1-intergin were located to tumour vessels, and additional stromal signals were observed for NG2 and α1-integrin in JAs. This study demonstrates a prominent collagen-type VI expression in JAs. The collagen-type VI may exert an important growth stimulus in this tumour.     

Siderophores as drug delivery agents: application of the “Trojan Horse” strategy

The outer membrane permeability barrier is an important resistance factor of bacterial pathogens. In combination with drug inactivating enzymes, target alteration and efflux, it can increase resistance dramatically. A strategy to overcome this membrane-mediated resistance is the misuse of bacterial transport systems. Most promising are those for iron transport. They are vital for virulence and survival of bacteria in the infected host, where iron depletion is a defense mechanism against invading pathogens. We synthesized biomimetic siderophores as shuttle vectors for active transport of antibiotics through the bacterial membrane. Structure activity relationship studies resulted in siderophore aminopenicillin conjugates that were highly active against Gram-negative pathogens which play a crucial role in destructive lung infections in cystic fibrosis patients and in severe nosocomial infections. The mechanism of action and the uptake of the compounds via specific iron siderophore transport routes were demonstrated. The novel conjugates were active against systemic Pseudomonas aeruginosa infections in mice with ED₅₀ values comparable to the quinolone ofloxacin and show low toxicity.     

1,4,9,10-Anthradiquinone as precursor for antitumor compounds

1,4,9,10-Anthradiquinone 5 was reacted with enamines 6 in the Nenitzescu reaction to yield unexpected 3,3a,6,12-tetrahydro-3a,7-dihydroxy-2-methyl-6,12-dioxo-naphtho[2,3-d]indol-1-carboxylates 8A. However, anthracycline-like naphtho-condensed 5-hydroxyindoles were not obtained from this diquinone. It yielded similar reaction products of the Nenitzescu reaction like other quinones activated by two electron-withdrawing groups. Furthermore, these new compounds 8A were found to constitute precursors for the synthesis of azonines. The conversion to dibenzoazonines 13 occurred in an unusual and up to now unknown way consisting of isomerization, ring opening, and re-closure. 2-Chloro-anthradiquinone 19 reacted with enamines 6 as vinylogeous acid chloride to pyrroloanthraquinone 20. No substitution of chlorine was observed. Naphtho-condensed indoles 26 were obtained by the reactions of unsubstituted 1,4-anthraquinone 25 with enamines 6 via the normal Nenitzescu route. Indoles 26 were converted to Mannich bases, reacting further to dimers by the Diels-Alder reaction of intermediate o-quinone methides. Most of the synthesized heterocycles were evaluated for their anticancer properties in the NCI's human-disease oriented in vitro anticancer screen. Particularly, carbinolamines 8A exhibited inhibitory activity of tumor cell growth and thus they constitute a new class of lead structures for anticancer drug design.     

RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function

The Cre/loxP recombinase system for performing conditional gene targeting experiments has been very useful in exploring genetic pathways that control both the development and function of pancreatic beta-cells. One particular line of transgenic mice (B6.Cg-Tg(Ins2-cre)25Mgn/J), commonly called RIP-Cre, in which expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used in at least 21 studies on at least 17 genes. In most of these studies inactivation of the gene of interest was associated with either glucose intolerance or frank diabetes. Experimental evidence has been gradually emerging to suggest that RIP-Cre mice alone display glucose intolerance. In this study experiments from three laboratories demonstrate that RIP-Cre mice, in the absence of genes targeted by loxP sites, are glucose intolerant, possibly due to impaired insulin secretion. In addition, we review the use of RIP-Cre mice and discuss possible molecular underpinnings and ramifications of our findings.     

Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing

Regulatory peptides represent a diverse group of messenger molecules. In insects, they are produced by endocrine cells as well as secretory neurones within the CNS. Many regulatory peptides are released as hormones into the haemolymph to regulate, for example, diuresis, heartbeat or ecdysis behaviour. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohaemal organs. We have performed a mass spectrometric characterization of the peptide complement of the main neurohaemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, we detected 23 different peptides of which five were unpredicted by previous genome screenings. We also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. Our results show that the peptidome of the neurohaemal organs is tagma-specific and does not change during metamorphosis. We also provide evidence for the first case of differential prohormone processing in Drosophila.     

Cation-exchange chromatography of monoclonal antibodies: Characterization of a novel stationary phase designed for production-scale purification

A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel^® SO₃⁻ (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behavior of mAbs onto Eshmuno™ S and Fractogel^® SO₃⁻ and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel^® SO₃⁻, indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel^® SO₃⁻ and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel^® SO₃⁻ or Toyopearl GigaCap S-650M.Key words: ion-exchange chromatography, dynamic binding capacity, tentacle surface modification, linear gradient elution, hcp removal     

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ～75% and single RyR2 opening frequency ～150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.