Toll-like receptors as transducer of inflammatory signals in glia: the astrocyte-microglia connection

In physiological conditions glia in the central nervous system (CNS) can produce and release protective factors such as anti-oxidant molecules and neurotrophic factors (Sofroniew et al., 2010). Events that impinge on CNS homeostatic balance can induce local inflammatory responses (Carson et al., 2006). Reactive glia can participate producing pro-inflammatory mediators such as chemokines, cytokines, purines and free radicals. Toll-like receptors (TLRs) are involved in injury responses of nervous system tissue and in neuropathic pain. Here we have investigated the cross-talk mechanisms between glial cells in the CNS making use of an in vitro cellular model, evaluating how glia respond to TLR agonists based on cytokine synthesis and release as well as TLR mRNA/protein expression as readouts. In order to analyze specific molecular parameters involved in the genesis and maintenance of inflammation, purified microglia and astrocyte-enriched cultures were generated from cerebral cortex of 1-2 day-old rat pups. For some experiments the enriched astrocyte cultures were purified by treatment with L-leucyl-L- leucine methyl ester (L-LME), which selectively depletes cultures of microglia. Activation of microglia and astrocytes (± L-LME) was achieved by treatment with lipopolysaccharide (LPS, TLR4 agonist); zymosan (TLR2 agonist) and poly(I:C) (TLR3 agonist) for 6 and 24 hours. Gene expression analysis (Real Time-polymerase chain reaction) revealed the ability of microglia to induce mRNA coding for interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In contrast, purified (nominally microglia-free) astrocyte cultures were not responsive to TLR agonists – unlike their astrocyte-enriched counterpart. Mediator production and release into the culture medium (analysed by ELISA) confirmed that microglia themselves respond to pathogenic stimuli. Utilizing flow-cytometric analysis we evaluated the expression of TLR receptors on the cell surface (TLR2/4) or in endosomal membranes (TLR3) after 1, 6 or 24 hours of stimulation with TLR agonists. Non-neuronal cell responsiveness to pathogenic stimuli is almost always linked to the production of inflammatory mediators. In this context we asked if the apparent inability of purified astrocytes to express a pro-inflammatory phenotype was dependent on the absence of the relevant TLR. Using confocal microscopy, stimulation with LPS conjugated with a fluorochrome showed the presence of TLR4 on the astrocyte cell surface. and Western blot analysis revealed the presence of the co-receptors MD2 and CD14. As consequence, purified astrocytes have been studied in flow cytometry to evaluate alteration in TLR protein expression. Moreover, we reconstituted the inflammatory profile in astrocyte cell cultures by adding fixed numbers of purified microglia (10% of contaminating cells final). Although the latter 'co-cultures' express pro-inflammatory cytokines after TLR agonist stimulation the absolute levels are inferior to those measured in enriched astrocytes (<5% of contaminating microglia. To further address the issue of whether microglial cell activation in the presence of astrocytes results from either physical interaction between cell membranes or chemical induction mediated by the release of mediator(s) into the culture medium, a “Transwell insert” system was used. The astrocyte/microglia co-culture paradigm described here may provide a useful starting point to elucidate the molecular mechanisms underlying astrocyte- and microglia-specific responses pertaining to, although not limited to, CNS inflammation, especially where TLR activation plays a role

Development of a Spatial Discount Task to Measure Impulsive Choices in Dogs

Impulsive choices reflect an individual\u2019s tendency to prefer a smaller immediate reward over a larger delayed one. Here, we have developed a behavioural test which can be easily applied to assess impulsive choices in dogs. Dogs were trained to associate one of two equidistant locations with a larger food amount when a smaller amount was presented in the other location, then the smaller amount was placed systematically closer to the dog. Choices of the smaller amount, as a function of distance, were considered a measure of the dog\u2019s tendency to make impulsive choices. All dogs (N = 48) passed the learning phase and completed the entire assessment in under 1 h. Choice of the smaller food amount increased as this was placed closer to the dog. Choices were independent from food motivation, past training, and speed of learning the training phase; supporting the specificity of the procedure. Females showed a higher probability of making impulsive choices, in agreement with analogue sex differences found in human and rodent studies, and supporting the external validity of our assessment. Overall, the findings support the practical applicability and represent a first indication of the validity of this method, making it suitable for investigations into impulsivity in dogs

Genetic testing for tetralogy of Fallot

Abstract Tetralogy of Fallot (ToF) combines congenital cardiac defects including ventricular septal defect, pulmonary stenosis, an overriding aorta and right ventricular hypertrophy. Clinical manifestation of this defect depends on the direction and volume of shunting of blood through the ventricular septal defect and the associated right ventricular and pulmonary artery pressures. ToF accounts for 3-5% of congenital heart defects or 0.28 cases every 1000 live births. ToF has autosomal dominant inheritance. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for pulmonary stenosis

Abstract Pulmonary stenosis (PS) is a congenital pulmonary valve malformation. It can be classified as valvular, subvalvular or supravalvular. Isolated forms of PS are rare. PS is associated with the development of massive pulmonary arterial dilatation. Patients with PS have a high consanguinity rate and the disorder is highly familial, which is why knowing the genetic aetiology of this defect is important. Prevalence is estimated at about 4/10,000 live births, and incidence at about 10% of all children with congenital heart defects. PS has prevalently autosomal dominant and rarely autosomal recessive inheritance. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for Marfan syndrome

Abstract Marfan syndrome (MFS) is an inherited connective tissue disorder caused by heterozygous mutations in the FBN 1 gene. Clinical manifestations of MFS include aortic dilatation and dissection, as well as cardiac valvular, ocular, skeletal and neurological manifestations. Prevalence varies from 6 to 20 per 100,000 individuals. Revised Ghent Nosology (2010) is used to establish a clinically based suspected diagnosis to be confirmed by molecular testing. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. Molecular testing is useful for diagnosis confirmation, as well as differential diagnosis, appropriate genetic counselling and access to clinical trials

Genetic testing for cerebral cavernous malformations

Abstract Cavernous cerebral malformations (CCM) are vascular malformations of the brain and spinal cord. CCM affect up to 0.5% of the general population, predisposing to headaches, seizures, cerebral hemorrhage and focal neurological deficit. CCM may be familial or sporadic. Familial forms have autosomal dominant inheritance. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Genetic testing for Marfan-like disorders

Abstract Marfan-like disorders are inherited conditions with features resembling Marfan syndrome but without a pathogenic variant in FBN1, and/or without a clinical diagnosis of Marfan syndrome according to the Revised Ghent criteria, and/or with a pathogenic variant in a different disease gene. Marfan-like disorders are clinically and genetically heterogeneous and have variable prognosis. They may have autosomal dominant or autosomal recessive patterns of inheritance. The prevalence of most Mar-fan-like disorders is unknown. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. Molecular testing is useful for diagnosis confirmation, as well as differential diagnosis, appropriate genetic counselling and access to clinical trials

Genetic testing for hereditary hemorrhagic telangiectasia

Abstract Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia characterized by telangiectases and arteriovenous malformations. These lesions cause bleeding, particularly in the nose, gastrointestinal tract and brain. HHT has incomplete penetrance, variable expressivity and genetic heterogeneity. De novo mutations associated with the onset of sporadic HHT have been reported. This Utility Gene Test was prepared on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System

Research background. In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. Experimental approach. We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. Results and conclusions. Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2\u2019-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. Novelty and scientific contribution. Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes

Effectiveness of third-class biologic treatment in crohn’s disease : A multi-center retrospective cohort study

Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Background: Multiple studies have described the effectiveness of ustekinumab (UST) and vedolizumab (VDZ) in patients with Crohn’s disease (CD) failing anti-Tumor necrosis factors (TNFs); however, the effectiveness of VDZ or UST as a third-class biologic has not yet been described. Aims and Methods: In this retrospective multicenter cohort study, we aimed to investigate the effectiveness of VDZ and UST as a third-class biologic in patients with CD. Results: Two-hundred and four patients were included; 156/204 (76%) patients received VDZ as a second-and UST as a third-class therapy (group A); the remaining 48/204 (24%) patients received UST as a second-and VDZ as a third-class therapy (group B). At week 16–22, 87/156 (55.5%) patients and 27/48 (56.2%) in groups A and B, respectively, responded to treatment (p = 0.9); 41/156 (26.2%) and 15/48 (31.2%) were in clinical remission (p = 0.5). At week 52; 89/103 (86%) patients and 25/29 (86.2%) of the patients with available data had responded to third-class treatment in groups A and B, respectively (p = 0.9); 31/103 (30%) and 47/29 (24.1%) were in clinical remission (p = 0.5). Conclusion: Third-class biological therapy was effective in more than half of the patients with CD. No differences in effectiveness were detected between the use of VDZ and UST as a third-class agent.Peer reviewe