Critical role for prokineticin 2 in CNS autoimmunity

Objective: To investigate the potential role of prokineticin 2 (PK2), a bioactive peptide involved in multiple biological functions including immune modulation, in CNS autoimmune demyelinating disease. Methods: We investigated the expression of PK2 in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), and in patients with relapsing-remitting MS. We evaluated the biological effects of PK2 on expression of EAE and on development of T-cell response against myelin by blocking PK2 in vivo with PK2 receptor antagonists. We treated with PK2 immune cells activated against myelin antigen to explore the immune-modulating effects of this peptide in vitro. Results: Pk2 messenger RNA was upregulated in spinal cord and lymph node cells (LNCs) of mice with EAE. PK2 protein was expressed in EAE inflammatory infiltrates and was increased in sera during EAE. In patients with relapsing-remitting MS, transcripts for PK2 were significantly increased in peripheral blood mononuclear cells compared with healthy controls, and PK2 serum concentrations were significantly higher. A PK2 receptor antagonist prevented or attenuated established EAE in chronic and relapsing-remitting models, reduced CNS inflammation and demyelination, and decreased the production of interferon (IFN)-γ and interleukin (IL)-17A cytokines in LNCs while increasing IL-10. PK2 in vitro increased IFN-γ and IL-17A and reduced IL-10 in splenocytes activated against myelin antigen. Conclusion: These data suggest that PK2 is a critical immune regulator in CNS autoimmune demyelination and may represent a new target for therapy

Glyphosate resistance by engineering the flavoenzyme glycine oxidase.

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and D-isomer of amino acids to yield the corresponding \u3b1-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the \u3b12-\u3b13 loop (comprising residues 50-60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide

Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation

The development of inflammatory diseases implies inactivation of regulatory T (Treg) cells through mechanisms that still are largely unknown. Here we showed that mast cells (MCs), an early source of inflammatory mediators, are able to counteract Treg inhibition over effector T cells. To gain insight into the molecules involved in their interplay, we set up an in vitro system in which all 3 cellular components were put in contact. Reversal of Treg suppression required T cell-derived interleukin-6 (IL-6) and the OX40/OX40L axis. In the presence of activated MCs, concomitant abundance of IL-6 and paucity of Th1/Th2 cytokines skewed Tregs and effector T cells into IL-17-producing T cells (Th17). In vivo analysis of lymph nodes hosting T-cell priming in experimental autoimmune encephalomyelitis revealed activated MCs, Tregs, and Th17 cells displaying tight spatial interactions, further supporting the occurrence of an MC-mediated inhibition of Treg suppression in the establishment of Th17-mediated inflammatory responses. © 2009 by The American Society of Hematology

Actual performance of mechanical ventilators in ICU: a multicentric quality control study.

Even if the performance of a given ventilator has been evaluated in the laboratory under very well controlled conditions, inappropriate maintenance and lack of long-term stability and accuracy of the ventilator sensors may lead to ventilation errors in actual clinical practice. The aim of this study was to evaluate the actual performances of ventilators during clinical routines. A resistance (7.69 cmH(2)O/L/s) - elastance (100 mL/cmH(2)O) test lung equipped with pressure, flow, and oxygen concentration sensors was connected to the Y-piece of all the mechanical ventilators available for patients in four intensive care units (ICUs; n = 66). Ventilators were set to volume-controlled ventilation with tidal volume = 600 mL, respiratory rate = 20 breaths/minute, positive end-expiratory pressure (PEEP) = 8 cmH(2)O, and oxygen fraction = 0.5. The signals from the sensors were recorded to compute the ventilation parameters. The average ± standard deviation and range (min-max) of the ventilatory parameters were the following: inspired tidal volume = 607 ± 36 (530-723) mL, expired tidal volume = 608 ± 36 (530-728) mL, peak pressure = 20.8 ± 2.3 (17.2-25.9) cmH(2)O, respiratory rate = 20.09 ± 0.35 (19.5-21.6) breaths/minute, PEEP = 8.43 ± 0.57 (7.26-10.8) cmH(2)O, oxygen fraction = 0.49 ± 0.014 (0.41-0.53). The more error-prone parameters were the ones related to the measure of flow. In several cases, the actual delivered mechanical ventilation was considerably different from the set one, suggesting the need for improving quality control procedures for these machines

Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-β

We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics

CD8+ T cells specific for cryptic apoptosis-associated epitopes exacerbate experimental autoimmune encephalomyelitis

The autoimmune immunopathology occurring in multiple sclerosis (MS) is sustained by myelin-specific and -nonspecific CD8(+) T cells. We have previously shown that, in MS, activated T cells undergoing apoptosis induce a CD8(+) T cell response directed against antigens that are unveiled during the apoptotic process, namely caspase-cleaved structural proteins such as non-muscle myosin and vimentin. Here, we have explored in vivo the development and the function of the immune responses to cryptic apoptosis-associated epitopes (AEs) in a well-established mouse model of MS, experimental autoimmune encephalomyelitis (EAE), through a combination of immunization approaches, multiparametric flow cytometry, and functional assays. First, we confirmed that this model recapitulated the main findings observed in MS patients, namely that apoptotic T cells and effector/memory AE-specific CD8(+) T cells accumulate in the central nervous system of mice with EAE, positively correlating with disease severity. Interestingly, we found that AE-specific CD8(+) T cells were present also in the lymphoid organs of unprimed mice, proliferated under peptide stimulation in vitro, but failed to respond to peptide immunization in vivo, suggesting a physiological control of this response. However, when mice were immunized with AEs along with EAE induction, AE-specific CD8(+) T cells with an effector/memory phenotype accumulated in the central nervous system, and the disease severity was exacerbated. In conclusion, we demonstrate that AE-specific autoimmunity may contribute to immunopathology in neuroinflammation

Can subtle changes in gene expression be consistently detected with different microarray platforms?

Background: The comparability of gene expression data generated with different microarray platforms is still a matter of concern. Here we address the performance and the overlap in the detection of differentially expressed genes for five different microarray platforms in a challenging biological context where differences in gene expression are few and subtle. Results: Gene expression profiles in the hippocampus of five wild-type and five transgenic δC-doublecortin-like kinase mice were evaluated with five microarray platforms: Applied Biosystems, Affymetrix, Agilent, Illumina, LGTC home-spotted arrays. Using a fixed false discovery rate of 10% we detected surprising differences between the number of differentially expressed genes per platform. Four genes were selected by ABI, 130 by Affymetrix, 3,051 by Agilent, 54 by Illumina, and 13 by LGTC. Two genes were found significantly differentially expressed by all platforms and the four genes identified by the ABI platform were found by at least three other platforms. Quantitative RT-PCR analysis confirmed 20 out of 28 of the genes detected by two or more platforms and 8 out of 15 of the genes detected by Agilent only. We observed improved correlations between platforms when ranking the genes based on the significance level than with a fixed statistical cut-off. We demonstrate significant overlap in the affected gene sets identified by the different platforms, although biological processes were represented by only partially overlapping sets of genes. Aberrances in GABA-ergic signalling in the transgenic mice were consistently found by all platforms. Conclusion: The different microarray platforms give partially complementary views on biological processes affected. Our data indicate that when analyzing samples with only subtle differences in gene expression the use of two different platforms might be more attractive than increasing the number of replicates. Commercial two-color platforms seem to have higher power for finding differentially expressed genes between groups with small differences in expression

Relative power and sample size analysis on gene expression profiling data

Background: With the increasing number of expression profiling technologies, researchers today are confronted with choosing the technology that has sufficient power with minimal sample size, in order to reduce cost and time. These depend on data variability, partly determined by sample type, preparation and processing. Objective measures that help experimental design, given own pilot data, are thus fundamental. Results: Relative power and sample size analysis were performed on two distinct data sets. The first set consisted of Affymetrix array data derived from a nutrigenomics experiment in which weak, intermediate and strong PPARα agonists were administered to wild-type and PPARα-null mice. Our analysis confirms the hierarchy of PPARα-activating compounds previously reported and the general idea that larger effect sizes positively contribute to the average power of the experiment. A simulation experiment was performed that mimicked the effect sizes seen in the first data set. The relative power was predicted but the estimates were slightly conservative. The second, more challenging, data set describes a microarray platform comparison study using hippocampal δC-doublecortin-like kinase transgenic mice that were compared to wild-type mice, which was combined with results from Solexa/Illumina deep sequencing runs. As expected, the choice of technology greatly influences the performance of the experiment. Solexa/Illumina deep sequencing has the highest overall power followed by the microarray platforms Agilent and Affymetrix. Interestingly, Solexa/Illumina deep sequencing displays comparable power across all intensity ranges, in contrast with microarray platforms that have decreased power in the low intensity range due to background noise. This means that deep sequencing technology is especially more powerful in dete

Occlusion of LTP-Like Plasticity in Human Primary Motor Cortex by Action Observation

Passive observation of motor actions induces cortical activity in the primary motor cortex (M1) of the onlooker, which could potentially contribute to motor learning. While recent studies report modulation of motor performance following action observation, the neurophysiological mechanism supporting these behavioral changes remains to be specifically defined. Here, we assessed whether the observation of a repetitive thumb movement – similarly to active motor practice – would inhibit subsequent long-term potentiation-like (LTP) plasticity induced by paired-associative stimulation (PAS). Before undergoing PAS, participants were asked to either 1) perform abductions of the right thumb as fast as possible; 2) passively observe someone else perform thumb abductions; or 3) passively observe a moving dot mimicking thumb movements. Motor evoked potentials (MEP) were used to assess cortical excitability before and after motor practice (or observation) and at two time points following PAS. Results show that, similarly to participants in the motor practice group, individuals observing repeated motor actions showed marked inhibition of PAS-induced LTP, while the “moving dot” group displayed the expected increase in MEP amplitude, despite differences in baseline excitability. Interestingly, LTP occlusion in the action-observation group was present even if no increase in cortical excitability or movement speed was observed following observation. These results suggest that mere observation of repeated hand actions is sufficient to induce LTP, despite the absence of motor learning