Concurrent short-term use of prednisolone with cyclosporine A accelerates pruritus reduction and improvement in clinical scoring in dogs with atopic dermatitis.

BACKGROUND: A randomized, unmasked, multicenter study was conducted to evaluate the rate of pruritus reduction and improvement in clinical scoring by cyclosporine A (5 mg/kg orally, once daily for 28 days) either alone (n = 25 dogs) or with concurrent prednisolone (1 mg/kg once daily for 7 days, followed by alternate dosing for 14 days; n = 23 dogs) for the treatment of atopic dermatitis in dogs. Dogs were included in the study after exclusion of other causes of pruritic dermatitis, and were assessed by dermatologists on days 0, 14 ± 1 and 28 ± 2. Assessments included: general physical examination, CADESI-03 lesion scoring, overall clinical response, evaluation of adverse events (AEs), body weight and clinical pathology (hematology, clinical chemistry and urinalysis). Owner assessments, including pruritus (visual analogue scale, VAS) and overall assessment of response were conducted every 3-4 days, either during visits to the clinic or at home. Owners reported AEs to the investigator throughout the study. RESULTS: By day 28 ± 2 both treatment groups resulted in a significant improvement of the atopic dermatitis. Both investigators and owners agreed that concurrent therapy resulted in a quicker improvement of the dogs 'overall' skin condition and of pruritus (significant reduction of pruritus by day 3-4, 72.8% improvement by day 14 ± 1), when compared to cyclosporine A alone (significant reduction of pruritus by day 7-8, 24.7% improvement by day 14 ± 1). CADESI-03 scores significantly improved in both groups by day 14 ± 1 onwards, and there were no significant differences in the scores between treatment groups at any time points. A total of 56 AEs (cyclosporine A alone = 34; concurrent therapy = 22) were reported in 33 dogs. No dogs died or stopped treatment due to an AE. The most commonly reported AEs in the cyclosporine A group were associated with the digestive tract, whilst systemic disorders were reported more frequently observed following concurrent therapy. Evaluation of body weight change and clinical pathology indices showed no overall clinically significant abnormalities. CONCLUSIONS: In dogs with atopic dermatitis, a short initiating course of prednisolone expedited the efficacy of cyclosporine A in resolving pruritus and associated clinical signs. The observed adverse events were consistent with those expected for the individual veterinary medicinal products.

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation.

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.

Selective regulation of nuclear orphan receptors 4A by adenosine receptor subtypes in human mast cells.

Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A(2A) (A(2A)AR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A(2B) and A(3)) and that A(2A)AR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A(2A)AR-silencing amplified NR4A induction by NECA. Interestingly, a similar A(2A)AR modulatory effect was observed on ERK1/2 phosphorylation because A(2A)AR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A(2A)AR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A(2A)AR activation, not only in the context of adenosine but of other mast cell activating stimuli as well.

A new PCR-based bioassay strategy for the detection of type A trichothecenes in food.

Type A trichothecenes (primarily T-2 and HT-2 toxins) are common fungal metabolites found in a wide range of grains and other field crops grown in temperate climatic zones. By acting as potent inhibitors of protein synthesis, T-2 and HT-2 exert adverse effects particularly against rapidly proliferating tissues, including the bone marrow, the immune system and epithelial cells. Based on toxicity studies in laboratory and farm animals, a temporary tolerable daily intake for the sum of T-2 and HT-2 has been issued in the European Union. However, exposure assessments suggest that the combined intake of these natural compounds exceeds in many cases the proposed threshold. To further protect the consumers, it is therefore necessary to screen a large number of food samples for parts per billion levels of both T-2 and HT-2. Towards that goal, we are the first to report that these two type A trichothecenes induce fast and high-amplitude transcriptional changes in cultured human breast cancer cells. This specific response involving marker gene inductions by more than 1000-fold has been exploited to develop a real-time PCR-based screening method that displays a limit of detection of 5 ng g(-1) for T-2 and 10 ng g(-1) for HT-2. The practicability of this bioassay is demonstrated by its application to the detection of type A trichothecenes in different food matrices.

Pleiotropic combinatorial transcriptomes of human breast cancer cells exposed to mixtures of dietary phytoestrogens.

Combinations of estrogen receptor agonists have been shown to exert more potent effects than single compounds in many single-endpoint bioassays. However, to our knowledge, it has never been tested how genome-wide expression programs are shaped by the interplay of multiple estrogenic stimuli. In view of the abundance of dietary phytoestrogens, we selected binary mixtures of these phytochemicals to determine their global impact using high-density DNA microarrays. MCF7 cells, a frequent in vitro model for molecular processes associated with breast cancer, were exposed to a sub-saturating concentration of coumestrol either alone or in combination with analogs that exhibit 1000-fold lower estrogen receptor activity. As expected, in the presence of coumestrol, the induction of many estrogen-sensitive genes was not further increased by the addition of resveratrol or enterolactone. However, it was surprising to find that these weak phytoestrogens, when combined with coumestrol in equal concentrations, were able to more than double the number of significantly regulated transcripts. Thus, phytoestrogens with low receptor affinity interact with other estrogenic agonists to generate more widespread expression fingerprints. This effect involving the number of susceptible transcripts instead of their amplitude of induction remains undetected if mixtures are evaluated with conventional bioassays.

Adenosine receptor modulation: potential implications in veterinary medicine.

Adenosine is a purine nucleoside whose concentration increases during inflammation and hypoxia and the many roles of this molecule are becoming better understood. Increased reactivity to adenosine of the airways of asthmatic but not of normal subjects underlines the role of adenosine in airway inflammation. The identification and pharmacological characterisation of different adenosine receptors have stimulated the search for subtype-specific ligands able to modulate the effects of this molecule in a directed way. Several compounds of different chemical classes have been identified as having potential drawbacks, including side effects resulting from the broad distribution of the receptors across the organism, have prevented clinical application. In this article, the effects of adenosine's different receptors and the intracellular signalling pathways are reviewed. The potential of adenosine receptor modulation as a therapeutic target for chronic airway inflammation is considered, taking equine recurrent airway disease and feline asthma as examples of naturally occurring airway obstructive diseases. Other potential applications for adenosine receptor modulation are also discussed. As the intrinsic molecular events of adenosine's mechanism of action become uncovered, new concrete therapeutic approaches will become available for the treatment of various conditions in veterinary medicine.

Global gene expression profiles induced by phytoestrogens in human breast cancer cells.

The nutritional intake of phytoestrogens seems to reduce the risk of breast cancer or other neoplastic diseases. However, these epidemiological findings remain controversial because low doses of phytoestrogens, achievable through soy-rich diets, stimulate the proliferation of estrogen-sensitive tumor cells. The question of whether such phytochemicals prevent cancer or rather pose additional health hazards prompted us to examine global gene expression programs induced by a typical soy product. After extraction from soymilk, phytoestrogens were deconjugated and processed through reverse- and normal-phase cartridges. The resulting mixture was used to treat human target cells that represent a common model system for mammary tumorigenesis. Analysis of mRNA on high-density microarrays revealed that soy phytoestrogens induce a genomic fingerprint that is indistinguishable from the transcriptional effects of the endogenous hormone 17beta-estradiol. Highly congruent responses were also observed by comparing the physiologic estradiol with daidzein, coumestrol, enterolactone, or resveratrol, each representing distinct phytoestrogen structures. More diverging transcriptional profiles were generated when an inducible promoter was used to reconstitute the expression of estrogen receptor beta (ERbeta). Therefore, phytoestrogens appear to mitigate estrogenic signaling in the presence of both ER subtypes but, in late-stage cancer cells lacking ERbeta, these phytochemicals contribute to a tumor-promoting transcriptional signature.

Expression profile of human cells in culture exposed to glycidamide, a reactive metabolite of the heat-induced food carcinogen acrylamide.

Recent findings of acrylamide in many common foods have sparked renewed interest in assessing human health hazards and the long-term risk associated with exposure to vinyl compounds. Acrylamide is tumorigenic at high doses in rodents and has been classified as a probable human carcinogen. However, cancer risk projections in the population remain problematic because the molecular pathogenesis of acrylamide at the low level of dietary uptake is not understood. In particular, the question of whether specific transcriptional responses may amplify or mitigate the known genotoxicity of acrylamide has never been examined. Here, we used high-density DNA microarrays and PCR validations to assess genome-wide messenger profiles induced by glycidamide, the more reactive metabolite of acrylamide. The expression changes resulting from glycidamide treatment of human epithelial cells are characterized by the induction of detoxification enzymes, several members of the glutathione system and antioxidant factors. Low-dose experiments indicate that the up-regulation of epoxide hydrolase 1 represents the most sensitive transcriptional biomarker of glycidamide exposure. At higher concentrations, glycidamide induces typical markers of tumor progression such as steroid hormone activators, positive regulators of nuclear factor-kappaB, growth stimulators and apoptosis inhibitors. Concomitantly, growth suppressors and cell adhesion molecules are down-regulated. The main implication of these findings for risk assessment is that low concentrations of glycidamide elicit cytoprotective reactions whereas transcriptional signatures associated with tumor progression may be expected only at doses that exceed the range of ordinary dietary exposures.

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA.

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.

Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair.

The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.

More than just strand breaks: the recognition of structural DNA discontinuities by DNA-dependent protein kinase catalytic subunit.

The DNA-dependent protein kinase (DNA-PK) is a trimeric factor originally identified as an enzyme that becomes activated upon incubation with DNA. Genetic defects in either the catalytic subunit (DNA-PK(CS)) or the two Ku components of DNA-PK result in immunodeficiency, radiosensitivity, and premature aging. This combined phenotype is generally attributed to the requirement for DNA-PK in the repair of DNA double strand breaks during various biological processes. However, recent studies revealed that DNA-PK(CS), a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to apoptotic cell death, telomere maintenance and other pathways of genome surveillance. These manifold functions of DNA-PK(CS) have been associated with an increasing number of protein interaction partners and phosphorylation targets. Here we review the DNA binding properties of DNA-PK(CS) and highlight its ability to interact with an astounding diversity of nucleic acid substrates. This survey indicates that the large catalytic subunit of DNA-PK functions as a sensor of not only broken DNA molecules, but of a wider spectrum of aberrant, unusual, or specialized structures that interrupt the standard double helical conformation of DNA.

Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair.

Using only a limited repertoire of recognition subunits, the nucleotide excision repair (NER) system is able to detect a nearly infinite variety of bulky DNA lesions. This extraordinary substrate versatility has generally been ascribed to an indirect readout mechanism, whereby particular distortions of the double helix, induced by a damaged nucleotide, provide the molecular determinants not only for lesion recognition but also for subsequent verification or demarcation processes. Here, we discuss the evidence in support of a bipartite mechanism of substrate discrimination that is initiated by the detection of thermodynamically unstable base pairs followed by direct localization of the lesion through an enzymatic proofreading activity. This bipartite discrimination mechanism is part of a dynamic reaction cycle that confers high levels of selectivity to avoid futile repair events on undamaged DNA and also protect the intact complementary strand from inappropriate cleavage.

Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions.

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PK(CS) (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PK(CS) associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin-streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PK(CS) with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PK(CS) binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PK(CS) adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PK(CS) to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

Age- and sex-dependent distribution of persistent organochlorine pollutants in urban foxes.

The colonization of urban and suburban habitats by red foxes (Vulpes vulpes) provides a novel sentinel species to monitor the spread of anthropogenic pollutants in densely populated human settlements. Here, red foxes were collected in the municipal territory of Zürich, Switzerland, and their perirenal adipose tissue was examined for persistent organochlorine residues. This pilot study revealed an unexpected pattern of contamination by polychlorinated biphenyls (PCBs), with significantly higher levels of the predominant congeners PCB-138, PCB-153, and PCB-180 in juvenile foxes relative to adult animals. Further data analysis demonstrated that the observed difference was attributable to an age-dependent reduction of PCB concentrations in females, whereas male foxes retained approximately the same PCB burden throughout their life span. A similar sex-related bias between population members has been observed, primarily in marine mammals. Interestingly, the reduction of organochlorine contents with progressive age is reminiscent of human studies, where an extensive maternal transfer of xenobiotics to the offspring has been shown to result in increased exposure levels of infants relative to adults. To our knowledge, this is the first example of an urban wildlife species that faithfully reflects the dynamic distribution of toxic contaminants in the corresponding human population. Suburban and urban foxes occupy habitats in close proximity to humans, depend on anthropogenic food supplies, are relatively long-lived and readily available for sampling, can be easily aged and sexed, have a limited home range, and, therefore, meet several important requirements to serve as a surrogate species for the assessment of toxic health hazards.