Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology

Pseudomonas aeruginosa Adaptation to Lungs of Cystic Fibrosis Patients Leads to Lowered Resistance to Phage and Protist Enemies

Pathogenic life styles can lead to highly specialized interactions with host species, potentially resulting in fitness trade-offs in other ecological contexts. Here we studied how adaptation of the environmentally transmitted bacterial pathogen, Pseudomonas aeruginosa, to cystic fibrosis (CF) patients affects its survival in the presence of natural phage (14/1, ΦKZ, PNM and PT7) and protist (Tetrahymena thermophila and Acanthamoebae polyphaga) enemies. We found that most of the bacteria isolated from relatively recently intermittently colonised patients (1-25 months), were innately phage-resistant and highly toxic for protists. In contrast, bacteria isolated from long time chronically infected patients (2-23 years), were less efficient in both resisting phages and killing protists. Moreover, chronic isolates showed reduced killing of wax moth larvae (Galleria mellonella) probably due to weaker in vitro growth and protease expression. These results suggest that P. aeruginosa long-term adaptation to CF-lungs could trade off with its survival in aquatic environmental reservoirs in the presence of microbial enemies, while lowered virulence could reduce pathogen opportunities to infect insect vectors; factors that are both likely to result in poorer environmental transmission. From an applied perspective, phage therapy could be useful against chronic P. aeruginosa lung infections that are often characterized by multidrug resistance: chronic isolates were least resistant to phages and their poor growth will likely slow down the emergence of beneficial resistance mutations

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, a-DsbA1 and a-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein a-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of a-DsbA1 for both dithiol oxidants(E. coli DsbA, 12%) and disulfide isomerases(E. coli DsbC, 14%) is similar. We therefore sought to establish whether a-DsbA1 is an oxidant or an isomerase based on functional activity. The purified a-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that a-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified a-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins. Copyright © 2008 Elsevier In

International multicentre observational study to assess the efficacy and safety of a 0·5 mg kg−1 per day starting dose of oral corticosteroids to treat bullous pemphigoid

BackgroundEuropean guidelines propose a 0 center dot 5 mg kg(-1) per day dose of oral prednisone as initial treatment for bullous pemphigoid (BP). We assessed the safety and efficacy of this regimen depending on BP extent and general condition of the patients.MethodsIn a prospective international study, we consecutively included all patients diagnosed with BP. Patients received a 0 center dot 5 mg kg(-1) per day dose of prednisone, which was then gradually tapered 15 days after disease control, with the aim of stopping prednisone or maintaining minimal treatment (0 center dot 1 mg kg(-1) per day) within 6 months after the start of treatment. The two coprimary endpoints were control of disease activity at day 21 and 1-year overall survival. Disease severity was assessed according to the Bullous Pemphigoid Disease Area Index (BPDAI) score.ResultsIn total, 198 patients were included between 2015 and 2017. The final analysis comprised 190 patients with a mean age of 80 center dot 9 (SD 9 center dot 1) years. Control of disease activity was achieved at day 21 in 119 patients [62 center dot 6%, 95% confidence interval (CI) 55 center dot 3-69.5]; 18 of 24 patients (75%, 95% CI 53 center dot 3-90 center dot 2), 75 of 110 patients (68 center dot 8%, 95% CI 59 center dot 2-77 center dot 3) and 26 of 56 patients (46.4%, 95% CI 33 center dot 0-60 center dot 3) had mild, moderate and severe BP, respectively (P = 0 center dot 0218). A total of 30 patients died during the study. The overall Kaplan-Meier 1-year survival was 82 center dot 6% (95% CI 76 center dot 3-87 center dot 4) corresponding to 90 center dot 9%, 83 center dot 0% and 80 center dot 0% rates in patients with mild, moderate and severe BP, respectively (P = 0 center dot 5). Thresholds of 49 points for BPDAI score and 70 points for Karnofsky score yielded maximal Youden index values with respect to disease control at day 21 and 1-year survival, respectively.ConclusionsA 0 center dot 5 mg kg(-1) per day dose of prednisone is a valuable therapeutic option in patients with mild or moderate BP whose general condition allows them to be autonomous.</p

Nested Regular Expressions can be Compiled to Small Deterministic Nested Word Automata

International audienceWe study the problem of whether regular expressions for nested words can be compiled to small deterministic nested word au-tomata (NWAs). In theory, we obtain a positive answer for small deter-ministic regular expressions for nested words. In practice of navigational path queries, nondeterministic NWAs are obtained for which NWA de-terminization explodes. We show that practical good solutions can be obtained by using stepwise hedge automata as intermediates

ATP-Dependent Infra-Slow (<0.1 Hz) Oscillations in Thalamic Networks

An increasing number of EEG and resting state fMRI studies in both humans and animals indicate that spontaneous low frequency fluctuations in cerebral activity at <0.1 Hz (infra-slow oscillations, ISOs) represent a fundamental component of brain functioning, being known to correlate with faster neuronal ensemble oscillations, regulate behavioural performance and influence seizure susceptibility. Although these oscillations have been commonly indicated to involve the thalamus their basic cellular mechanisms remain poorly understood. Here we show that various nuclei in the dorsal thalamus in vitro can express a robust ISO at ∼0.005–0.1 Hz that is greatly facilitated by activating metabotropic glutamate receptors (mGluRs) and/or Ach receptors (AchRs). This ISO is a neuronal population phenomenon which modulates faster gap junction (GJ)-dependent network oscillations, and can underlie epileptic activity when AchRs or mGluRs are stimulated excessively. In individual thalamocortical neurons the ISO is primarily shaped by rhythmic, long-lasting hyperpolarizing potentials which reflect the activation of A1 receptors, by ATP-derived adenosine, and subsequent opening of Ba2+-sensitive K+ channels. We argue that this ISO has a likely non-neuronal origin and may contribute to shaping ISOs in the intact brain

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes

An intrinsic vasopressin system in the olfactory bulb is involved in social recognition

Many peptides, when released as chemical messengers within the brain, have powerful influences on complex behaviours. Most strikingly, vasopressin and oxytocin, once thought of as circulating hormones whose actions were confined to peripheral organs, are now known to be released in the brain where they play fundamentally important roles in social behaviours1. In humans, disruptions of these peptide systems have been linked to several neurobehavioural disorders, including Prader-Willi syndrome, affective disorders, and obsessive-compulsive disorder, and polymorphisms of the vasopressin V1a receptor have been linked to autism2,3. Here we report that the rat olfactory bulb contains a large population of interneurones which express vasopressin, that blocking the actions of vasopressin in the olfactory bulb impairs the social recognition abilities of rats, and that vasopressin agonists and antagonists can modulate the processing of information by olfactory bulb neurones. The findings indicate that social information is processed in part by a vasopressin system intrinsic to the olfactory system