TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

HGF Mediates the Anti-inflammatory Effects of PRP on Injured Tendons

Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2. © 2013 Zhang et al

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Molecular biology of histidine decarboxylase and prostaglandin receptors

Histamine and prostaglandins (PGs) play a variety of physiological roles as autacoids, which function in the vicinity of their sources and maintain local homeostasis in the body. They stimulate target cells by acting on their specific receptors, which are coupled to trimeric G proteins. For the precise understanding of the physiological roles of histamine and PGs, it is necessary to clarify the molecular mechanisms involved in their synthesis as well as their receptor-mediated responses. We cloned the cDNAs for mouse l-histidine decarboxylase (HDC) and 6 mouse prostanoid receptors (4 PGE2 receptors, PGF receptor, and PGI receptor). We then characterized the expression patterns and functions of these genes. Furthermore, we established gene-targeted mouse strains for HDC and PG receptors to explore the novel pathophysiological roles of histamine and PGs. We have here summarized our research, which should contribute to progress in the molecular biology of HDC and PG receptors

Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

© 2010 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 5 (2010): e8537, doi:10.1371/journal.pone.0008537.Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW; Walter A. and Hope Noyes Smith, and a National Science Foundation Graduate Research Fellowship to KEW

Contrast-enhanced CMR in patients after percutaneous closure of the left atrial appendage: A pilot study

<p>Abstract</p> <p>Background</p> <p>To evaluate the feasibility and value of first-pass contrast-enhanced dynamic and post-contrast 3D CMR in patients after transcatheter occlusion of left atrial appendage (LAA) to identify incorrect placement and persistent leaks.</p> <p>Methods</p> <p>7 patients with different occluder systems (n = 4 PLAATO; n = 2 Watchman; n = 1 ACP) underwent 2 contrast-enhanced (Gd-DOTA) CMR sequences (2D TrueFISP first-pass perfusion and 3D-TurboFLASH) to assess localization, artifact size and potential leaks of the devices. Perfusion CMR was analyzed visually and semi-quantitatively to identify potential leaks.</p> <p>Results</p> <p>All occluders were positioned within the LAA. The ACP occluder presented the most extensive artifact size. Visual assessment revealed a residual perfusion of the LAA apex in 4 cases using first-pass perfusion and 3D-TurboFLASH indicating a suboptimal LAA occlusion.</p> <p>By assessing signal-to-time-curves the cases with a visually detected leak showed a 9-fold higher signal-peak in the LAA apex (567 ± 120% increase from baseline signal) than those without a leak (61 ± 22%; p < 0.03). In contrast, the signal increase in LAA proximal to the occluder showed no difference (leak 481 ± 201% vs. no leak 478 ± 125%; p = 0.48).</p> <p>Conclusion</p> <p>This CMR pilot study provides valuable non-invasive information in patients after transcatheter occlusion of the LAA to identify correct placement and potential leaks. We recommend incorporating CMR in future clinical studies to evaluate new device types.</p

Impaired neonatal macrophage phagocytosis is not explained by overproduction of prostaglandin E2

<p>Abstract</p> <p>Background</p> <p>Neonates and young infants manifest increased susceptibility to bacterial, viral and fungal lung infections. Previous work has identified a role for eicosanoids in mediating host defense functions of macrophages. This study examines the relationship between alveolar macrophage (AM) host defense and production of lipid mediators during the neonatal period compared to adult AMs.</p> <p>Methods</p> <p>AMs were harvested from young (day 7 and day 14) and adult (~10 week) rats. The functionality of these cells was assessed by examining their ability to phagocytose opsonized targets, produce cytokines, eicosanoids and intracellular cAMP measured by enzyme immunoassays, and gene expression of proteins, enzymes and receptors essential for eicosanoid generation and phagocytosis measured by real time RT-PCR.</p> <p>Results</p> <p>AMs from young animals (day 7 and 14) were defective in their ability to phagocytose opsonized targets and produce tumor necrosis factor (TNF)- α. In addition, young AMs produce more prostaglandin (PG) E₂, a suppressor of host defense, and less leukotriene (LT) B₄, a promoter of host defense. Young AMs express higher levels of enzymes responsible for the production of PGE₂and LTB₄; however, there was no change in the expression of E prostanoid (EP) receptors or LT receptors. Despite the similar EP profiles, young AMs are more responsive to PGE₂as evidenced by their increased production of the important second messenger, cyclic AMP. In addition, young AMs express higher levels of PDE3B and lower levels of PDE4C compared to adult AMs. However, even though the young AMs produced a skewed eicosanoid profile, neither the inhibition of PGE₂by aspirin nor the addition of exogenous LTB₄rescued the defective opsonized phagocytosis. Examination of a receptor responsible for mediating opsonized phagocytosis showed a significant decrease in the gene expression levels of the Fcgamma receptor in young (day 7) AMs compared to adult AMs.</p> <p>Conclusion</p> <p>These results suggest that elevated production of PGE₂and decreased production of LTB₄do not contribute to impaired opsonized macrophage phagocytosis and highlight an important difference between young and adult AMs.</p

Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study

BACKGROUND: Recent studies show that inflammatory processes may contribute to neuropathic pain. Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for production of prostanoids, which may sensitise sensory neurones via the EP1 receptor. We have recently reported that while macrophages infiltrate injured nerves within days of injury, they express increased Cox-2-immunoreactivity (Cox-2-IR) from 2 to 3 weeks after injury. We have now investigated the time course of EP1 and Cox-2 changes in injured human nerves and dorsal root ganglia (DRG), and the chronic constriction nerve injury (CCI) model in the rat. METHODS: Tissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis. RESULTS: EP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord. CONCLUSION: Different stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain