Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2).

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

Usage of international standards for integrating extramural monitoring and personal health device data into medical information infrastructure.

Integrating extramural measured devices data into medical information systems is becoming more and more attractive for integrated medical care. A lot of devices already have the ability to transfer measured data to mobile devices or computers and a few systems offer submitting data to a centralized information database or information system. Unfortunately, all of these devices use proprietary protocols and processes which makes integration into other systems a major problem. To address this problem the Healthy Interoperability project has been created with the objective of creating a framework for transferring health data based on international standards. The paper outlines how the framework architecture takes full advantage from the definitions of the international standards ISO 11073, HL7, IHE and CEN 13606. Even the definition of the user profiles and the security framework is based on standards from ETSI, ISO and CEN. By using these standards the framework can also perfectly be used for intramural communication.

Stromal Cells in the Pathogenesis of Inflammatory Bowel Disease.

Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma.

Flip-flop-induced relaxation of bending energy: implications for membrane remodeling.

Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids--they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system.

BACKGROUND: Current efforts to develop stem cell therapy as a novel treatment for neurointestinal diseases are limited by the unavailability of a model system to study cell transplantation in the human intestine. We propose that xenograft models support enteric nervous system (ENS) development in the fetal human intestine when transplanted into mice subcutaneously or intra-abdominally. METHODS: Fetal human small and large intestine were grafted onto the small intestinal mesentery and into the subcutaneous tissue of immunodeficient mice for up to 4 months. Intestinal cytoarchitecture and ENS development were studied using immunohistochemistry. KEY RESULTS: In both abdominal and subcutaneous grafts, the intestine developed normally with formation of mature epithelial and mesenchymal layers. The ENS was patterned in two ganglionated plexuses containing enteric neurons and glia, including cholinergic and nitrergic neuronal subtypes. c-Kit-immunoreactive interstitial cells of Cajal were present in the gut wall. CONCLUSIONS & INFERENCES: Abdominal xenografts represent a novel model that supports the growth and development of fetal human intestine. This in vivo approach will be a useful method to study maturation of the ENS, the pathophysiology of neurointestinal diseases, and the long-term survival and functional differentiation of neuronal stem cells for the treatment of enteric neuropathies.

Inhibition of the effects of adenosine on force of contraction and the slow calcium inward current by pertussis toxin is associated with myocardial lesions.

The effects of adenosine and the adenosine receptor agonist (-)-N(6)-phenyl-isopropyladenosine (PIA) in the presence of isoprenaline on isometric force of contraction and calcium dependent slow action potentials were studied in papillary muscles from guinea pigs pretreated with pertussis toxin and control guinea pigs. Hearts from guinea pigs treated in the same way with pertussis toxin or solvent alone underwent histological examination. For comparison, hearts from isoprenaline treated guinea pigs were also studied. Pertussis toxin specifically inactivates guanine nucleotide binding proteins (N proteins) involved in transmembrane signal transduction in many receptor systems (for example, adenosine receptors, m-cholinoceptors, and and alpha 2 adrenoceptors). In papillary muscles from control guinea pigs adenosine and PIA in the presence of isoprenaline produced a negative inotropic effect and inhibited the maximal rate of depolarisation of slow calcium dependent action potentials in potassium depolarised papillary muscles. After pretreatment with pertussis toxin the inhibitory effects both on force of contraction and on the maximal rate of depolarisation of adenosine and PIA were abolished. Treatment with pertussis toxin produced disseminated myocardial necrosis and a disseminated cellular calcium overload evidenced by glyoxal-2-bis-hydroxyanil (GBHA) staining. Similar lesions (for example, myocardial necrosis and cellular calcium overload) were also observed after treatment with isoprenaline. In controls neither myocardial necrosis nor cellular calcium overload was found. It is concluded that pertussis toxin sensitive N proteins are involved in the inhibitory effects of adenosine and PIA on force of contraction and on slow calcium inward current during beta adrenergic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of shuttle vectors for Bacillus subtilis and Escherichia coli.

A series of shuttle vectors for Bacillus subtilis and Escherichia coli was developed. These are derived from one basic construct composed of parts of the Gram+ plasmid pUB110 and the Gram- plasmid pBR322. They contain multiple cloning sites flanked by transcriptional terminators. In one plasmid, a vegetative B. subtilis promoter drives transcription of inserted genes. For the construction of operon and gene fusions, the cat of pUB112 and the lacZ gene of E. coli were employed as reporter genes. With these vectors, cloning and expression of genes as well as probing of regulatory signals can be performed in B. subtilis and E. coli.

Sequence and analysis of the replication region of the Staphylococcus xylosus plasmid pSX267.

The region of the 29.5-kb plasmid pSX267 from Staphylococcus xylosus DSM 20267 that is required for autonomous replication in staphylococci was isolated on a 1.8-kb DNA fragment. The sequence analysis of the fragment yielded two open reading frames, repA and orf2, encoding proteins of 37.2 and 13.2 kDa, respectively. The deduced amino acid sequence of repA showed similarity to the replication initiator protein of plasmid pAD1 from Enterococcus faecalis, to two proteins of unknown function encoded by the E. faecalis plasmid pCF10 and the lactobacillus helveticus plasmid pLJ1, and surprisingly to the mouse interferon-response element binding factor 1. The repA gene of pSX267 is indispensable for replication suggesting that it encodes the replication initiator protein of pSX267. Introduction of a frameshift mutation into repA or deletion of 26 codons at its 3'-end resulted in nonreplicative plasmids. Removal of sequences required for repA expression also abolished replication. Complementation experiments with repA in trans identified the ori of pSX267 within the repA coding region. The second orf, which is located downstream of repA, could be deleted without affecting plasmid replication. It seems to be a nonfunctional remainder of a rolling circle plasmid of the pC194/pUB110 family, since its deduced amino acid sequence resembles the pC194/pUB110-type replication proteins. The minimal replicon of pSX267 defined so far comprises 1255 bp. Besides repA, no other plasmid-encoded gene is required to mediate replication in staphylococci.

Expression of a chloramphenicol-resistance determinant carried on hybrid plasmids in gram-positive and gram-negative bacteria.

To analyse the control of chloramphenicol (Cm) resistance conferred by the Staphylococcus aureus plasmid pUB112, a detailed restriction map of this plasmid has been constructed, and the position and orientation of the cat gene have been determined. An MboI restriction fragment carrying the entire cat gene of pUB112 was then cloned in another S. aureus plasmid, the kanamycin (Km) resistance vector pUB110. Depending on the orientation of the incorporated cat fragment, the level of Cm resistance varied dramatically in Bacillus subtilis cells. This effect could not be eliminated by deleting parts of the vector DNA, and only the introduction of a transcription termination signal led to orientation-independent Cm resistance. One such construct was further developed to yield a shuttle vector, replicating both in Escherichia coli and B. subtilis. Using this vector the expression of incorporated genes can be determined in both Gram-positive and Gram-negative bacteria. By in vitro transcription experiments using pUB110 DNA linearized with various restriction endonucleases as template, two pUB110 promoters could be localized and their orientations determined: one promoter controls a gene whose function is unknown, the other regulates the transcription of the KmR gene.

Synthesis, pharmacological characterization, and quantitative structure-activity relationship analyses of 3,7,9,9-tetraalkylbispidines: derivatives with specific bradycardic activity.

A series of 3,7,9,9-tetraalkyl-3,7-diazabicyclo[3.3.1]nonane derivatives (bispidines) was synthesized and identified as potential antiischemic agents. Pharmacological experiments in vitro as well as in vivo are described, and the results are listed. For selection of those compounds fitting best to the desired profile of a specific bradycardic antianginal agent--decrease in heart rate without affecting contractility and blood pressure--these results were scored and ranked. Quantitative structure--activity relationship (QSAR) analyses were performed and discussed a posteriori by means of Hansch, nonelementary discriminant and factor analysis to get insight into the molecular features determining the biological profile. Highly significant equations were obtained, indicating hydrophobic and steric effects. Both pharmacological ranking and QSAR considerations showed compound 6 as the optimum within the structural class under investigation. Compound 6 (tedisamil, KC8857) has been selected as the most promising compound and was chosen for further pharmacological and clinical investigations as an antiischemic drug.

Effects of alpha-adrenoceptor stimulation with phenylephrine in the presence of propranolol on force of contraction, slow inward current and cyclic AMP content in the bovine heart.

The mechanism of the cyclic AMP-independent positive inotropic effect of cardiac alpha-adrenoceptor stimulation was studied by analyzing the effects of phenylephrine on force of contraction, calcium-dependent slow action potentials and the slow inward current (Isi) in bovine ventricular trabeculae. The preparations were electrically driven at 0.3 Hz in the presence of propranolol 1 mumol 1(-1). Phenylephrine increased the force of contraction in a concentration-dependent manner (maximum about 200% of control at 30 mumol 1(-1). The effect was surmountably antagonized by phentolamine. The positive inotropic effect of phenylephrine was accompanied by a concentration-dependent increase in time to peak force and occurred without any detectable increase in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. The positive inotropic effect of phenylephrine was accompanied by an increase in action potential duration both at 20% and 90% repolarization. Calcium-dependent slow action potentials were also prolonged by phenylephrine and there was a distinct increase in the maximal rate of depolarization (dV/dtmax) of these slow potentials. These effects were also completely reversible on washing and surmountably blocked by phentolamine. However, the increase in dV/dtmax was smaller than that of isoprenaline in concentrations producing similar inotropic effects. Voltage-clamp experiments with the single sucrose-gap method showed that the phenylephrine-induced increase in force of contraction was associated not only with an increase in peak slow calcium inward current, Isi max, but also with a delay in the inactivation of Isi. Outward currents were not detectably altered by phenylephrine. It is concluded that the alpha-adrenoceptor mediated, cyclic AMP-independent positive inotropic effects of phenylephrine in bovine cardiac muscle are associated with an increase in slow inward current. Additionally, the amount of calcium influx during excitation is probably increased by a delay in the inactivation of Isi. Both effects can explain the phenylephrine-produced prolongation of the action potential, and probably contribute to the positive inotropic effect of alpha-adrenoceptor stimulation. However, as the effect on dV/dtmax is smaller than that of isoprenaline, other (still unknown) mechanisms may also be involved.

Adenosine inhibits the positive inotropic effect of 3-isobutyl-1-methylxanthine in papillary muscles without effect on cyclic AMP or cyclic GMP.

1 Adenosine and the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA) produced a small positive and negative inotropic effect, respectively, in isolated electrically driven papillary muscles of guinea-pigs. 2 Adenosine (100 mumol l-1) had no effect on cyclic AMP or cyclic GMP content. PIA (100 mumol l-1) slightly increased cyclic AMP. 3 In the presence of 3-isobutyl-1-methylxanthine (IBMX; 60 mumol l-1), which increased force of contraction 2 fold, adenosine and PIA exerted strong negative inotropic effects. PIA was more potent than adenosine (mean IC25 2.1 and 168 mumol -1, respectively). 4 In contrast, the nucleosides did not affect the increase in force of contraction produced by elevating extracellular Ca2+ concentration. 5 The IBMX-antagonistic effects of adenosine and PIA were not accompanied by modification of the IBMX-induced increase in cyclic AMP and cyclic GMP. 6 The effects of adenosine and PIA on force of contraction were accompanied by a partial reversal of the IBMX-induced increase in the maximal rate of depolarization of slow action potentials. 7 It is concluded that adenosine and PIA are able to attenuate the positive inotropic effect of a phosphodiesterase inhibitor. This effect is unlikely to be due to a reduction of the IBMX-induced increase in cyclic AMP content. It is conceivably due to an inhibition of the stimulant action of cyclic AMP on slow Ca2+ channels leading to the reduction of the slow inward current which in turn reduces force of contraction.

Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus.

A system for high-efficiency gene replacement in Staphylococcus carnosus and Staphylococcus xylosus has been developed, that is based on temperature-sensitive Escherichia coli-Staphylococcus shuttle vectors for fragment delivery and erythromycin resistance cassettes to facilitate selection of genomic copies of disrupted genes. The approach was tested by constructing a phosphotransferase-deficient mutant of S. carnosus and an S. xylosus mutant strain unable to utilize sucrose. Allelic replacements were observed at rather high frequencies, ranging from approximately 10% for the ptsI gene in S. carnosus up to 50% for the scrB gene in S. xylosus. These differences most likely reflect the length of homology rather than strain-specific variations in recombination efficiencies. Apart from the staphylococcal species tested in this study, the system appears to be applicable in other staphylococci.

Identification of the serine acetyltransferase gene of Staphylococcus xylosus.

A transposon Tn917-induced mutant strain of Staphylococcus xylosus was isolated that required exogenous cysteine for growth. The transposon was found to reside within a gene, designated cysE, encoding a protein of 216 amino acids with a high level of similarity to bacterial serine acetyltransferases. The cysE::Tn917 mutant completely lost serine acetyltransferase activity, which is easily detectable in the wild-type strain. In addition, the mutant strain could no longer grow in minimal medium without cysteine. Therefore, the cysE gene product is essential for the de novo synthesis of cysteine via O-acetyl-L-serine in S. xylosus. The cysE gene is surrounded by genes encoding glutamyl-tRNA synthetase (gltX) and cysteinyl-tRNA synthetase (cysS), as deduced from sequence comparisons. The genetic organisation in S. xylosus, gltX-cysE-cysS, is identical to that found in Bacillus subtilis and Bacillus stearothermophilus.

A novel translocation (17;19)(p13;p13) in a patient with acute myelomonocytic leukemia.

We report on a patient with acute myeloid leukemia (AML M4) and a so far unrecorded translocation (17;19). The leukemia transformed from a myeloproliferative disorder (MPD) and showed a progressive fatal course. Following transformation, all leukemic cells showed an apparently balanced translocation (17;19)(p13;p13). The breakpoint regions harbor genes such as TP53 (17p13) and E2A, ENL, or LYL1 (19p13), which could be relevant in leukemogenesis. We suspect that the translocation (17;19)(p13;p13) may be a prognostic factor for transformation from chronic MPD to acute leukemia.

Alpha-adrenoceptor-mediated positive inotropic effect of phenylephrine in isolated human ventricular myocardium.

In isolated human ventricular myocardium the alpha-adrenoceptor agonist phenylephrine had a positive inotropic effect in preparations from 9 of 14 patients. This effect was seen in the presence of the beta-adrenoceptor-blocking agent propranolol but was nearly abolished by the alpha-adrenoceptor blocking agent prazosin. In contrast to the beta-adrenoceptor-mediated positive inotropic effect, the effect of phenylephrine was accompanied by a prolongation of the isometric contraction. The results suggest that alpha-adrenoceptors exist in human ventricular myocardium.

Effects on transmembrane action potential, slow inward current and force of contraction in ventricular cardiac muscle of BRL 31660, a new antiarrhythmic drug with class I and class IV activity.

The effects of 1-30 mumol l-1 BRL 31660 on transmembrane action potential and force of contraction were investigated in guinea-pig electrically driven papillary muscles. Lidocaine was studied for comparison. BRL 31660 depressed Vmax of the action potential without changing the resting potential, decreased the force of contraction and decreased the action potential duration. Similar effects were obtained with lidocaine. BRL 31660 inhibited the recovery of Vmax from inactivation, the time constant of which was estimated to be about 1,100 ms in the presence of 10 mumol l-1 BRL 31660. The depressive effect on Vmax was particularly pronounced at low (less negative) membrane potentials. BRL 31660 can thus be classified as a class I antiarrhythmic agent of the lidocaine type. Additional voltage-clamp experiments in cow ventricular trabeculae provided evidence that BRL 31660 also depressed the slow inward current at concentrations similar to those producing the effects on the transmembrane action potential. BRL 31660 thus exerted an additional class IV action. This effect was not shared by lidocaine. It is concluded that BRL 31660 is a new antiarrhythmic agent which depresses both the fast and slow inward current at similar concentrations. The dual effects of BRL 31660 conceivably contribute to its antiarrhythmic activity, but the clinical relevance of these results remains to be elucidated.

The positive inotropic effect of phenylephrine in the presence of propranolol. Increase in time to peak force and in relaxation time without increase in c-AMP.

The effects of phenylephrine on the shape of the contraction curve and on the cyclic adenosine 3',5'-monophosphate (c-AMP) content were studied in electrically driven (frequency 0.2 Hz) cat papillary muscles. All experiments were done in the presence of 1 micron propranolol in order to minimize interference from beta-adrenoceptors. 1. Phenylephrine increased the force of contraction in a concentration-dependent manner. Maximal effects (about 200% of control) occurred at 30 micron phenylephrine. 2. The positive inotropic effect (PIE) of phenylephrine was antagonized by phentolamine. Phentolamine, 5 micron, produced a parallel shift of the concentration-response curve for the PIE of phenylephrine by about two log units to the right. 3. The PIE of 30 micron phenylephrine occurred without any detectable increase in the c-AMP levels of the preparations. 4. The PIE of 30 micron phenylephrine developed about three times more slowly than the PIE of an equieffective concentration of isoprenaline. 5. The PIE of phenylephrine was accompanied by significant, concentration-dependent increases in both time to peak force and relaxation time. 6. It is concluded that the PIE of phenylephrine in the presence of propranolol is mediated mainly by a stimulation of alpha-adrenoceptors. It is unlikely to be related to an increase in c-AMP. With respect to time course and influence on the shape of the contraction curve it is qualitatively different from the effects of beta-adrenoceptor stimulation. These data are taken to support the hypothesis that the mechanical effects of alpha- and beta-adrenoceptor stimulating agents on the heart are produced by different mechanisms.

Role of guanine nucleotide-binding protein in the regulation by adenosine of cardiac potassium conductance and force of contraction. Evaluation with pertussis toxin.

In atrial cardiac preparations adenosine exerts a receptor-mediated negative inotropic effect due to an increased potassium conductance. Pretreatment of guinea pigs with pertussis toxin abolished the negative inotropic and action potential shortening effect of adenosine and the adenosine analogue (-)-N6-phenylisopropyladenosine (PIA). As pertussis toxin specifically inactivates guanine nucleotide-binding proteins involved in the signal transfer from receptor binding to specific cell functions, it is concluded that a guanine nucleotide-binding protein is involved in the regulation of the receptor-mediated change in potassium conductance and force of contraction.