BACKGROUND:Dog allergens are a common cause of allergic sensitisation and trigger respiratory symptoms worldwide. However, clinical evidence regarding dog immunotherapy is limited. Therefore, the aim of this study was to analyse the immunomodulatory properties of a new allergoid from dog dander, thereby deepening the understanding of the molecular mechanisms involved in the reestablishment of the tolerogenic response.METHODS:Three independent batches of dog dander native and allergoid allergen extracts were manufactured and characterised. Allergenic profiles were analysed by the identification of all dog allergens and quantification of the major allergens Can f 1 and Can f 5. The allergenicity profile of the allergoid was studied using biological potency and basophil activation tests. In vitro immunomodulatory parameters was evaluated as the capacity of the allergoid to induce IgG antibodies that block IgE binding to the allergen and cytokine promotion (IFN-γ, IL-4, IL-6, IL-10, IL-13, and TNF-α) in PBMCs from allergic donors.RESULTS:The presence of all dog allergens, including Can f 1 and Can f 5, was confirmed in both types of extracts. The new allergoid showed a low IgE binding capacity, which significantly affected the activation of effector cells, such as basophils. The IgG antibodies induced by the allergoid in rabbits blocked human IgE binding epitopes on the dog native extract and induced Th1 and Treg responses by increasing IFN-γ and IL-10 levels in PBMCs from allergic donors.CONCLUSION:This new dog dander allergoid containing Can f 1 and Can f 5 showed a low capacity to bind IgE and to activate basophils in dog allergic patients. Furthermore, it showed potent activation of Th1 mediators and induction of tolerance through Treg activation. This allergoid could offer a safer profile than the native extract and could be an effective immunotherapy treatment for dog allergic patients.

Type II (proteic) toxin-antitoxin (TA) operons are widely spread in bacteria and archaea. They are organized as operons in which, usually, the antitoxin gene precedes the cognate toxin gene. The antitoxin generally acts as a transcriptional self-repressor, whereas the toxin acts as a co-repressor, both proteins constituting a harmless complex. When bacteria encounter a stressful environment, TAs are triggered. The antitoxin protein is unstable and will be degraded by host proteases, releasing the free toxin to halt essential processes. The result is a cessation of cell growth or even death. Because of their ubiquity and the essential processes targeted, TAs have been proposed as good candidates for development of novel antimicrobials. We discuss here the possible druggability of TAs as antivirals and antibacterials, with focus on the potentials and the challenges that their use may find in the 'real' world. We present strategies to develop TAs as antibacterials in view of novel technologies, such as the use of very small molecules (fragments) as inhibitors of protein-protein interactions. Appropriate fragments could disrupt the T:A interfaces leading to the release of the targeted TA pair. Possible ways of delivery and formulation of Tas are also discussed.

This chapter focuses on the proteic toxin-antitoxin (TA) systems. TA systems function as vitally important regulatory systems in bacteria and represent ideal targets for the development of novel antibiotic therapeutic agents. A broad mechanistic understanding of TA systems at physiological, biochemical, biophysical, and structural levels provides the scientific framework needed both for rational drug design and for elegant selection schemes using large pools of compounds. The proteins of chromosome-encoded TA systems (relBE, yefM-yoeB, and dinJ-yafQ) from gram-negative bacteria, namely, CcdA-CcdB, Phd-Doc, ParD-ParE, YefM-YoeB, and one system from a plasmid from a G+ bacterium, have been studied in vitro with respect to their properties in solution and binding to DNA. The chapter summarizes the knowledge accumulated on these proteins. Pathogenic bacteria are subjected to an enormous selective pressure because of the indiscriminate overuse and misuse of broad-spectrum antibiotics. The recognition of the importance of protein-protein interactions within the cell has led to their investigation as targets for novel inhibitors. Here, the approaches that can be used for screening of inhibitors of protein-protein interactions are highlighted by recent research on the TA systems. The chapter focuses on two resonance energy transfer techniques, namely, fluorescent resonance energy transfer (FRET) and, especially, bioluminescence resonance energy transfer (BRET), since they have demonstrated to be highly useful for studying interactions between two proteins that have been shown to form complexes.

Background:Clinical trials in ulcerative colitis (UC) rely on certain parameters to evaluate responses that are highly subjective or of low sensitivity. Here, using a select group of genes, we tested the accuracy of gene expression analysis as a biomarker of clinical, endoscopic, and histologic improvements. Methods:Intestinal biopsies were obtained from UC patients included in two cohorts. Cohort 1 was used to select for genes whose expression was modulated in active (vs. inactive) UC. Cohort 2 included patients recruited in a phase II study receiving placebo, mesalazine, or dersalazine sodium for 4 weeks. The expression of 44 genes identified in Cohort 1 was assessed at weeks 0 and 4, and was then correlated with biomarkers, as well as with clinical, endoscopic, and histologic scores. Results:Significant changes in the expression of 31 of the 44 genes tested were detected in Cohort 2 at week 4. Gene expression (&Dgr;Ct) significantly correlated with the total Mayo score, C-reactive protein (CRP), and fecal calprotectin. The number of genes significantly regulated at week 4 was highly associated with histologic and endoscopic responses. Logistic regression analysis identified four separate genes (IFITM1, ITGB2, IL1R2, IL2RA) whose relative change was independently associated with endoscopic remission with high specificity and sensitivity. Conclusions:Change in the expression of a select set of genes can serve as an early biomarker, one with high specificity and sensitivity to clinical, endoscopic, and histologic responses. This could represent a new tool for identifying early response to treatment in mild to moderately active UC patients.

BACKGROUND AND PURPOSE Dersalazine sodium (DS) is a new chemical entity formed by combining, through an azo bond, a potent platelet activating factor (PAF) antagonist (UR‐12715) with 5‐aminosalicylic acid (5‐ASA). DS has been demonstrated to have anti‐inflammatory effects on trinitrobenzene sulphonic acid (TNBS)‐induced colitis in rats and recently in UC patients in phase II PoC. There is Increasing evidence that Th17 cells have an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to further characterize the anti‐inflammatory effects of DS.EXPERIMENTAL APPROACH Effect of DS (10 or 30 mg·kg−1 b.i.d.) on TNBS‐induced colitis in rats was studied after 2 and 7 days with special focus on inflammatory mediators. Additionally, its anti‐inflammatory properties were analysed in two different models of dextran sodium sulphate (DSS)‐induced colitis, BALB/c and C57BL/6 mice, the latter being dependent on IL‐17.KEY RESULTS DS, when administered for 7 days, showed intestinal anti‐inflammatory effects in TNBS‐induced colitis; these effects were observed both macroscopically and through the profile of inflammatory mediators (TNF, IL‐1β, IL‐6 and IL‐17). Although the 2 day treatment with DS did not induce intestinal anti‐inflammatory effects, it was sufficient to reduce the enhanced IL‐17 expression. DS showed beneficial effects on DSS‐induced colitis in C57BL/6 mice and reduced colonic pro‐inflammatory cytokines IL‐1β, IL‐6 and IL‐17. In contrast, it did not exert intestinal anti‐inflammatory effects on DSS‐induced colitis in BALB/c mice.CONCLUSIONS AND IMPLICATIONS DS exerts intestinal anti‐inflammatory activity in different rodent models of colitis through down‐regulation of IL‐17 expression.

The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin–antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded TA loci, the labile ε antitoxin binds to free ζ toxin and neutralizes it. When the ζ toxin is freed from the ε antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged ζ toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the ε·ζ interaction can be considered as an attractive antimicrobial agent. Gene ε was fused to luc (Luc-ε antitoxin) and ζ to the gfp gene (ζ-GFP). Luc-ε or ε antitoxin neutralizes the toxic effect of the ζ or ζ-GFP toxin. In the absence of the antitoxin, free ζ or ζ-GFP triggers a reversible loss of cell proliferation, but the ζK46A-GFP variant fails to block growth. Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). To develop the proper controls, molecular dynamics studies were used to predict that the Asp18 and/or Glu22 residues might be relevant for ε·ζ interaction. Luc-ε efficiently transfers the excited energy to the fluorescent acceptor molecule (ζ-GFP or ζK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from ζ (D18A) affects Luc-ε·ζD18A K46A-GFP interaction. In this study, we validate the hypothesis that it is possible to disrupt a TA module and offer a novel and unexploited targets to fight against antibiotic-resistant strains.

Background and purpose: Highly selective M 3 muscarinic receptor antagonists may represent a better treatment for overactive bladder syndrome, diminishing side effects. Cardiac side effects of non‐selective antimuscarinics have been associated with activity at M 2 receptors as these receptors are mainly responsible for muscarinic receptor‐dependent bradycardia. We have investigated a novel antimuscarinic, SVT‐40776, highly selective for M 3 over M 2 receptors (Ki = 0.19 nmol·L −1 for M 3 receptor affinity). This study reports the functional activity of SVT‐40776 in the bladder, relative to its activity in atria. Experimental approach: In vitro and ex vivo (oral dosing) inhibition of mouse detrusor and atrial contractile responses to carbachol were used to study the functional activity of SVT‐40776. The in vivo efficacy of SVT‐40776 was characterized by suppression of isovolumetric spontaneous bladder contractions in anaesthetized guinea pigs after intravenous administration. Key results: SVT‐40776 was the most potent in inhibiting carbachol‐induced bladder contractions of the anti‐cholinergic agents tested, without affecting atrial contractions over the same range of concentrations. SVT‐40776 exhibited the highest urinary versus cardiac selectivity (199‐fold). In the guinea pig in vivo model, SVT‐40776 inhibited 25% of spontaneous bladder contractions at a very low dose (6.97 µg·kg −1 i.v), without affecting arterial blood pressure. Conclusions and implications: SVT‐40776 is a potent inhibitor of M 3 receptor‐related detrusor contractile activity. The absence of effects on isolated atria preparations represents an interesting characteristic and suggests that SVT‐40776 may lack unwanted cardiac effects; a feature especially relevant in a compound intended to treat mainly elderly patients. British Journal of Pharmacology (2009) doi:10.1111/j.1476‐5381.2008.00082.x

The aim of the present study was to evaluate the inmunomodulatory effects of UR-1505, a new salicylate derivative, on the T helper (Th)2/humoral response produced during dextran sodium sulfate (DSS)-induced rat colitis. In the in vitro studies, UR-1505 (300 microM) inhibited both the production of interleukin (IL)-10 and IL-5 in concanavalin A (Con A)-activated splenocytes and the production of immunoglobulin (Ig) G and IgA by B-lymphocytes. However, in contrast to the in vitro results, the administration of UR-1505 (10 and 30 mg/kg per day) to rats with established DSS-colitis enhanced both IL-10 and IgA production, whereas it inhibited IgG production, thus ameliorating the intestinal inflammation.