Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.     

The Role of Cognitive Styles and Sociodemographic Characteristics in Consumer Perceptions and Attitudes Toward Nonhuman Animal Welfare

Given the increasing importance of exploring consumers' concerns about the welfare of farmed animals, a survey questionnaire was designed to investigate the role of cognitive styles along with sociodemographic characteristics in consumers' perceptions about nonhuman animal welfare (AW) and their willingness to pay for animal-friendly products. The results revealed that the survey respondents were concerned about AW and had negative perceptions of the way animals were treated. They showed positive attitudes toward some actions to be taken for improving AW and strongly agreed to pay more for animal-friendly products. Consistent with previous studies, results revealed significant associations between sociodemographics and concern toward AW. However, some observed differences were highlighted by cognitive styles rather than by sociodemographic characteristics. These results indicate a significant link between cognitive styles and perceptions and attitudes toward AW, which may outweigh previously found sociodemographic differences and fuel the contemporary debate on AW.     

SYBR green real time-polymerase chain reaction as a rapid and alternative assay for the efficient identification of all existing Escherichia coli biotypes approved directly in wastewater samples

Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm‐blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses SYBR green real time‐polymerase chain reaction (RT‐PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT‐PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT‐PCR analysis can encourage the testing of a higher number of samples. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1106–1113, 2012     

Insights into the mechanism of oxidative deamination catalyzed by DOPA decarboxylase

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5'-phosphate (PLP) enzyme DOPA decarboxylase (DDC) was here investigated. Either wild-type or Y332F DDC variant is able to perform such oxidation toward aromatic amines or aromatic l-amino acids, respectively, without the aid of any cofactor related to oxygen chemistry. Oxidative deamination produces, in equivalent amounts, a carbonyl compound and ammonia, accompanied by dioxygen consumption in a 1:2 molar ratio with respect to the products. Kinetic studies either in the pre-steady or in the steady state, together with HPLC analyses of reaction mixtures under varying experimental conditions, revealed that a ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to PLP when the substrate is consumed. Rapid-mixing chemical quench studies provide evidence that the ketimine is indeed an intermediate formed during the first catalytic cycle. Moreover, superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. On this basis, a mechanism of oxidative deamination consistent with the present data is proposed. Furthermore, the catalytic properties of the T246A DDC mutant together with those previously obtained with H192Q mutant allow us to propose that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines.     

Reactions of human liver peroxisomal alanine:glyoxylate aminotransferase with beta-chloro-L-alanine and L-cysteine: spectroscopic and kinetic analysis

In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-l-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with l-alanine. On the other hand, during the reaction of AGT with l-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of l-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of l-cysteine is proposed. l-cysteine partitions between these two reactions with a ratio of ~2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of l-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of l-alanine and l-serine, respectively. Conversely, l-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of l-alanine and l-serine, respectively. This appears to be consistent with the calculated interaction energies of the l-cysteine, l-alanine and l-serine docked at the active site of AGT.     

Lack of Toll IL-1R8 exacerbates Th17 cell responses in fungal infection

TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8(-/-) mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8(-/-) mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-beta in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI(-/-) mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.     

Antiviral activity of the long chain pentraxin PTX3 against influenza viruses

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3-/- mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.     

IL-17 and therapeutic kynurenines in pathogenic inflammation to fungi

Largely viewed as proinflammatory, innate responses combine with adaptive immunity to generate the most effective form of antifungal resistance, and T cells exercise feedback control over diverse effects of inflammation on infection. Some degree of inflammation is required for protection, particularly in mucosal tissues, during the transitional response occurring between the rapid innate and slower adaptive response. However, progressive inflammation worsens disease and ultimately prevents pathogen eradication. IDO, tryptophan catabolites ("kynurenines"), and regulatory T cells help to tame overzealous and exaggerated inflammatory responses. In this context, IL-23 and the Th17 pathway, which down-regulate tryptophan catabolism, may instead favor pathology and serve to accommodate the seemingly paradoxical association of chronic inflammation with fungal persistence. Recent data support a view in which IL-23/IL-17 antagonistic strategies, including the administration of synthetic kynurenines, could represent a new means of harnessing progressive or potentially harmful inflammation.