Comparative transcriptome analysis of equine alveolar macrophages

Reasons for performing study: Alveolar macrophages (AMs) are the first line of defence against pathogens in the lungs of all mammalian species and thus may constitute appropriate therapeutic target cells in the treatment and prevention of opportunistic airway infections. Therefore, acquiring a better understanding of equine macrophage biology is of paramount importance in addressing this issue in relation to the horse. Objectives: To compare the transcriptome of equine AMs with that of equine peritoneal macrophages (PMs) and to investigate the effect of lipopolysaccharide (LPS) on equine AM. Study design: Gene expression study of equine AMs. Methods: Cells from both bronchoalveolar and peritoneal lavage fluid were isolated from systemically healthy horses that had been submitted to euthanasia. Cells were cryopreserved. RNA was extracted and comparative microarray analyses were performed in AMs and PMs, and in AMs treated and untreated with LPS. Comparisons with published data derived from human AM studies were made, with particular focus on LPS-induced inflammatory status. Results: The comparison between AMs and PMs revealed the differential basal expression of 451 genes. Gene expression analysis revealed an alternative (M2) macrophage polarisation profile in AMs and a hybrid macrophage activation profile in PMs, a phenomenon potentially attributable to a degree of induced endotoxin tolerance. The gene expression profile of equine AMs following LPS stimulation revealed significant changes in the expression of 240 genes, including well-known upregulated inflammatory genes. This LPS-induced gene expression profile of equine AMs more closely resembles that of human rather than murine macrophages. Conclusions: This study improves current understanding of equine macrophage biology. These data suggest that the horse may represent a suitable animal model for the study of human macrophage-associated lung inflammation and data derived from human macrophage studies may have significant relevance to the horse

Lipopolysaccharide promotes Drp1-dependent mitochondrial fission and associated inflammatory responses in macrophages

Mitochondria have a multitude of functions, including energy generation and cell signaling. Recent evidence suggests that mitochondrial dynamics (i.e. the balance between mitochondrial fission and fusion) also regulate immune functions. Here, we reveal that lipopolysaccharide (LPS) stimulation increases mitochondrial numbers in mouse bone marrow‐derived macrophages (BMMs) and human monocyte‐derived macrophages. In BMMs, this response requires Toll‐like receptor 4 (Tlr4) and the TLR adaptor protein myeloid differentiation primary response 88 (MyD88) but is independent of mitochondrial biogenesis. Consistent with this phenomenon being a consequence of mitochondrial fission, the dynamin‐related protein 1 (Drp1) GTPase that promotes mitochondrial fission is enriched on mitochondria in LPS‐activated macrophages and is required for the LPS‐mediated increase in mitochondrial numbers in both BMMs and mouse embryonic fibroblasts. Pharmacological agents that skew toward mitochondrial fusion also abrogated this response. LPS triggered acute Drp1 phosphorylation at serine 635 (S635), followed by sustained Drp1 dephosphorylation at serine 656 (S656), in BMMs. LPS‐induced S656 dephosphorylation was abrogated in MyD88‐deficient BMMs, suggesting that this post‐translational modification is particularly important for Tlr4‐inducible fission. Pharmacological or genetic targeting of Tlr4‐inducible fission had selective effects on inflammatory mediator production, with LPS‐inducible mitochondrial fission promoting the expression and/or secretion of a subset of inflammatory mediators in BMMs and mouse embryonic fibroblasts. Thus, triggering of Tlr4 results in MyD88‐dependent activation of Drp1, leading to inducible mitochondrial fission and subsequent inflammatory responses in macrophages.Ronan Kapetanovic, Syeda Farhana Afroz, Divya Ramnath, Grace MEP Lawrence, Takashi Okada, James EB Curson, Jost de Bruin, David P Fairlie, Kate Schroder, Justin C St John, Antje Blumenthal, Matthew J Swee

Bacterial resistance to arsenic protects against protist killing

Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli ars operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use arsenic to poison bacterial cells in the phagosome, similar to their use of copper. In response to copper and arsenic poisoning by protists, there is selection for acquisition of arsenic and copper resistance genes in the bacterial prey to avoid killing. In agreement with this hypothesis, both copper and arsenic resistance determinants are widespread in many bacterial taxa and environments, and they are often found together on plasmids. A role for heavy metals and arsenic in the ancient predator–prey relationship between protists and bacteria could explain the widespread presence of metal resistance determinants in pristine environments

Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion.

IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion

Early changes in bone mineral density measured by digital X-ray radiogrammetry predict up to 20 years radiological outcome in rheumatoid arthritis

ABSTRACT: INTRODUCTION: Change in bone mineral density (BMD) in the hand, as evaluated by digital X-ray radiogrammetry (DXR) of the II-IV metacarpal bones, has been suggested to predict future joint damage in rheumatoid arthritis (RA). This study's objective was to investigate if DXR-BMD loss early in the disease predicts development of joint damage in RA patients followed for up to 20 years. METHODS: 183 patients (115 women and 68 men) with early RA (mean disease duration 11 months) included from 1985 to 1989 were followed prospectively (the Lund early RA cohort). Clinical and functional measures were assessed yearly. Joint damage was evaluated according to the Larsen score on radiographs of hands and feet taken in years 0 to 5, 10, 15 and 20. These radiographs were digitized and BMD of the II-IV metacarpal bones was evaluated by DXR (Sectra, Linkoping. Sweden). Early DXR-BMD change rate (bone loss) per year calculated from the first 2 radiographs taken on average 9 months apart (SD 4.8) were available for 135 patients. Mean values of right and left hand were used. RESULTS: Mean early DXR-BMD loss during the first year calculated was -0.023 g/cm2 (SD 0.025). Patients with marked bone loss, i.e. early DXR-BMD loss above the median for the group, had significantly worse progression of joint damage at all examinations during the 20-year period. CONCLUSIONS: Early DXR-BMD progression rate predicted development of joint damage evaluated according to Larsen at year one and further onwards up to 20 years in this cohort of early RA patients

A chicken bioreactor for efficient production of functional cytokines

The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications

Antidepressant and antipsychotic use in an Italian pediatric population

<p>Abstract</p> <p>Background</p> <p>The safety and effectiveness of psychotropic drug use in the paediatric population is widely debated, in particular because of the lack of data concerning long term effects.</p> <p>In Italy the prevalence of psychotropic drug prescriptions increased in the early 2000s and decreased afterwards. In such a context, a study with the aim to estimate the incidence and prevalence of psychotropic drug prescription in the paediatric population and to describe diagnostic and therapeutic approaches was performed.</p> <p>Methods</p> <p>The study population was composed of 76,000 youths less than 18 years and living in the area covered by the local health unit of Verona, Italy. The data source was the Verona local health unit's administrative prescription database. Prevalence and incidence of antidepressant and/or antipsychotic drug prescriptions in the 2004-2008 period were estimated. Children and adolescents receiving antidepressant and/or antipsychotic drug prescriptions between 1 January 2005 and 31 December 2006 were identified and questionnaires were sent to the prescribers with the aim to collect data concerning diagnostic and therapeutic approaches, and care strategies.</p> <p>Results</p> <p>The prevalence of psychotropic drug prescriptions did not change in the 2004-2008 period, while incidence slightly increased (from 7.0 in 2005 to 8.3 per 10,000 in 2008). Between 1 January 2005 and 31 December 2006, 111 youths received at least one psychotropic drug prescription, 91 of whom received antidepressants. Only 28 patients attended child and adolescent psychiatry services. Information concerning diagnostic and therapeutic approaches, and care strategies was collected for 52 patients (47%). Anxiety-depressive syndrome and attention disorders were the diseases for which psychotropic drugs were most commonly prescribed. In all, 75% youths also received psychological support and 20% were prescribed drugs for 2 or more years.</p> <p>Conclusions</p> <p>Despite the low drug prescription prevalence, the finding that most children were not cared for by child and adolescent psychiatric services is of concern and calls for a systematic, continuous monitoring of psychopharmacological treatments.</p

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS

Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders