Basal protein phosphatase 2A activity restrains cytokine expression: Role for MAPKs and tristetraprolin

PP2A is a master controller of multiple inflammatory signaling pathways. It is a target in asthma; however the molecular mechanisms by which PP2A controls inflammation warrant further investigation. In A549 lung epithelial cells in vitro we show that inhibition of basal PP2A activity by okadaic acid (OA) releases restraint on MAPKs and thereby increases MAPK-mediated pro-asthmatic cytokines, including IL-6 and IL-8. Notably, PP2A inhibition also impacts on the anti-inflammatory protein - tristetraprolin (TTP), a destabilizing RNA binding protein regulated at multiple levels by p38 MAPK. Although PP2A inhibition increases TTP mRNA expression, resultant TTP protein builds up in the hyperphosphorylated inactive form. Thus, when PP2A activity is repressed, pro-inflammatory cytokines increase and anti-inflammatory proteins are rendered inactive. Importantly, these effects can be reversed by the PP2A activators FTY720 and AAL(s), or more specifically by overexpression of the PP2A catalytic subunit (PP2A-C). Moreover, PP2A plays an important role in cytokine expression in cells stimulated with TNFα; as inhibition of PP2A with OA or PP2A-C siRNA results in significant increases in cytokine production. Collectively, these data reveal the molecular mechanisms of PP2A regulation and highlight the potential of boosting the power of endogenous phosphatases as novel anti-inflammatory strategies to combat asthmatic inflammation

Activating protein phosphatase 2A (PP2A) enhances tristetraprolin (TTP) anti-inflammatory function in A549 lung epithelial cells

© 2016. Chronic respiratory diseases are driven by inflammation, but some clinical conditions (severe asthma, COPD) are refractory to conventional anti-inflammatory therapies. Thus, novel anti-inflammatory strategies are necessary. The mRNA destabilizing protein, tristetraprolin (TTP), is an anti-inflammatory molecule that functions to induce mRNA decay of cytokines that drive pathogenesis of respiratory disorders. TTP is regulated by phosphorylation and protein phosphatase 2A (PP2A) is responsible for dephosphorylating (and hence activating) TTP, amongst other targets. PP2A is activated by small molecules, FTY720 and AAL(S), and in this study we examine whether these compounds repress cytokine production in a cellular model of airway inflammation using A549 lung epithelial cells stimulated with tumor necrosis factor α (TNFα) in vitro. PP2A activators significantly increase TNFα-induced PP2A activity and inhibit mRNA expression and protein secretion of interleukin 8 (IL-8) and IL-6; two key pro-inflammatory cytokines implicated in respiratory disease and TTP targets. The effect of PP2A activators is not via an increase in TNFα-induced TTP mRNA expression; instead we demonstrate a link between PP2A activation and TTP anti-inflammatory function by showing that specific knockdown of TTP with siRNA reversed the repression of TNFα-induced IL-8 and IL-6 mRNA expression and protein secretion by FTY720. Therefore we propose that PP2A activators affect the dynamic equilibrium regulating TTP; shifting the equilibrium from phosphorylated (inactive) towards unphosphorylated (active) but unstable TTP. PP2A activators boost the anti-inflammatory function of TTP and have implications for future pharmacotherapeutic strategies to combat inflammation in respiratory disease

The phosphorylated form of FTY720 activates PP2A, represses inflammation and is devoid of S1P agonism in A549 lung epithelial cells

Protein phosphatase 2A (PP2A) activity can be enhanced pharmacologically by PP2A-activating drugs (PADs). The sphingosine analog FTY720 is the best known PAD and we have shown that FTY720 represses production of pro-inflammatory cytokines responsible for respiratory disease pathogenesis. Whether its phosphorylated form, FTY720-P, also enhances PP2A activity independently of the sphingosine 1-phosphate (S1P) pathway was unknown. Herein, we show that FTY720-P enhances TNF-induced PP2A phosphatase activity and significantly represses TNF-induced interleukin 6 (IL-6) and IL-8 mRNA expression and protein secretion from A549 lung epithelial cells. Comparing FTY720 and FTY720-P with S1P, we show that unlike S1P, the sphingosine analogs do not induce cytokine production on their own. In fact, FTY720 and FTY720-P significantly repress S1P-induced IL-6 and IL-8 production. We then examined their impact on expression of cyclooxygenase 2 (COX-2) and resultant prostaglandin E2 (PGE2) production. S1P did not increase production of this pro-inflammatory enzyme because COX-2 mRNA gene expression is NF-κB-dependent, and unlike TNF, S1P did not activate NF-κB. However, TNF-induced COX-2 mRNA expression and PGE2 secretion is repressed by FTY720 and FTY720-P. Hence, FTY720-P enhances PP2A activity and that PADs can repress production of pro-inflammatory cytokines and enzymes in A549 lung epithelial cells in a manner devoid of S1P agonism

Islet isolation assessment in man and large animals

Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity and in vitro and in vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 μ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason, in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers. © 1990 Casa Editrice il Ponte

Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina

BACKGROUND: Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection. METHODS: The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology. RESULTS: The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive. The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates. Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found. Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative. CONCLUSIONS: Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay

Enhancing tristetraprolin activity reduces the severity of cigarette smoke-induced experimental chronic obstructive pulmonary disease

© 2019 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc. Objective: Chronic obstructive pulmonary disease (COPD) is a progressive disease that causes significant mortality and morbidity worldwide and is primarily caused by the inhalation of cigarette smoke (CS). Lack of effective treatments for COPD means there is an urgent need to identify new therapeutic strategies for the underlying mechanisms of pathogenesis. Tristetraprolin (TTP) encoded by the Zfp36 gene is an anti-inflammatory protein that induces mRNA decay, especially of transcripts encoding inflammatory cytokines, including those implicated in COPD. Methods: Here, we identify a novel protective role for TTP in CS-induced experimental COPD using Zfp36aa/aa mice, a genetically modified mouse strain in which endogenous TTP cannot be phosphorylated, rendering it constitutively active as an mRNA-destabilising factor. TTP wild-type (Zfp36+/+) and Zfp36aa/aa active C57BL/6J mice were exposed to CS for four days or eight weeks, and the impact on acute inflammatory responses or chronic features of COPD, respectively, was assessed. Results: After four days of CS exposure, Zfp36aa/aa mice had reduced numbers of airway neutrophils and lymphocytes and mRNA expression levels of cytokines compared to wild-type controls. After eight weeks, Zfp36aa/aa mice had reduced pulmonary inflammation, airway remodelling and emphysema-like alveolar enlargement, and lung function was improved. We then used pharmacological treatments in vivo (protein phosphatase 2A activator, AAL(S), and the proteasome inhibitor, bortezomib) to promote the activation and stabilisation of TTP and show that hallmark features of CS-induced experimental COPD were ameliorated. Conclusion: Collectively, our study provides the first evidence for the therapeutic potential of inducing TTP as a treatment for COPD

The role of asymmetric interactions on the effect of habitat destruction in mutualistic networks

Plant-pollinator mutualistic networks are asymmetric in their interactions: specialist plants are pollinated by generalist animals, while generalist plants are pollinated by a broad involving specialists and generalists. It has been suggested that this asymmetric ---or disassortative--- assemblage could play an important role in determining the equal susceptibility of specialist and generalist plants under habitat destruction. At the core of the argument lies the observation that specialist plants, otherwise candidates to extinction, could cope with the disruption thanks to their interaction with generalist pollinators. We present a theoretical framework that supports this thesis. We analyze a dynamical model of a system of mutualistic plants and pollinators, subject to the destruction of their habitat. We analyze and compare two families of interaction topologies, ranging from highly assortative to highly disassortative ones, as well as real pollination networks. We found that several features observed in natural systems are predicted by the mathematical model. First, there is a tendency to increase the asymmetry of the network as a result of the extinctions. Second, an entropy measure of the differential susceptibility to extinction of specialist and generalist species show that they tend to balance when the network is disassortative. Finally, the disappearance of links in the network, as a result of extinctions, shows that specialist plants preserve more connections than the corresponding plants in an assortative system, enabling them to resist the disruption.Comment: 14 pages, 7 figure

Vascular disease and vascular risk factors in relation to motor features and cognition in early Parkinson's disease

OBJECTIVE: The purpose of this study was to examine the relationship between vascular disease (and vascular risk factors), cognition and motor phenotype in Parkinson's disease (PD). METHODS: Recently diagnosed PD cases were enrolled in a multicenter prospective observational longitudinal cohort study. Montreal cognitive assessment (normal >23, mild cognitive impairment 22 to 23 or lower but without functional impairment, and dementia 21 or less with functional impairment) and Movement Disorder Society Unified PD Rating Scale part 3 (UPDRS 3) scores were analyzed in relation to a history of vascular events and risk factors. RESULTS: In 1759 PD cases, mean age 67.5 (standard deviation 9.3) years, mean disease duration 1.3 (standard deviation 0.9) years, 65.2% were men, 4.7% had a history of prior stroke or transient ischemic attack, and 12.5% had cardiac disease (angina, myocardial infarction, heart failure). In cases without a history of vascular disease, hypertension was recorded in 30.4%, high cholesterol 27.3%, obesity 20.7%, diabetes 7.2%, and cigarette smoking in 4.6%. Patients with prior stroke or transient ischemic attack were more likely to have cognitive impairment (42% vs 25%) and postural instability gait difficulty (53.5% vs 39.5%), but these findings were not significant after adjustment for age, sex, and disease duration (P = .075). The presence of more than 2 vascular risks was associated with worse UPDRS 3 motor scores (beta coefficient 4.05, 95% confidence interval 1.48, 6.61, p = .002) and with cognitive impairment (ordinal odds ratio 2.24, 95% confidence interval 1.34, 3.74, p = .002). In 842 patients (47.8%) with structural brain imaging, white matter leukoaraiosis, but not lacunar or territorial infarction, was associated with impaired cognition (p = .006) and postural instability gait difficulty (p = .010). CONCLUSION: Vascular comorbidity is significantly associated with cognitive and gait impairment in patients with early PD, which may have prognostic and treatment implications. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society

Rare variants analysis of cutaneous malignant melanoma genes in Parkinson's disease

A shared genetic susceptibility between cutaneous malignant melanoma (CMM) and Parkinson's disease (PD) has been suggested. We investigated this by assessing the contribution of rare variants in genes involved in CMM to PD risk. We studied rare variation across 29 CMM risk genes using high-quality genotype data in 6875 PD cases and 6065 controls and sought to replicate findings using whole-exome sequencing data from a second independent cohort totaling 1255 PD cases and 473 controls. No statistically significant enrichment of rare variants across all genes, per gene, or for any individual variant was detected in either cohort. There were nonsignificant trends toward different carrier frequencies between PD cases and controls, under different inheritance models, in the following CMM risk genes: BAP1, DCC, ERBB4, KIT, MAPK2, MITF, PTEN, and TP53. The very rare TYR p.V275F variant, which is a pathogenic allele for recessive albinism, was more common in PD cases than controls in 3 independent cohorts. Tyrosinase, encoded by TYR, is the rate-limiting enzyme for the production of neuromelanin, and has a role in the production of dopamine. These results suggest a possible role for another gene in the dopamine-biosynthetic pathway in susceptibility to neurodegenerative Parkinsonism, but further studies in larger PD cohorts are needed to accurately determine the role of these genes/variants in disease pathogenesis

Which circulating antioxidant vitamins are confounded by socioeconomic deprivation? The MIDSPAN family study

<p><b>Background:</b> Antioxidant vitamins are often described as having “independent” associations with risk of cancer, cardiovascular disease (CVD) and mortality. We aimed to compare to what extent a range of antioxidant vitamins and carotenoids are associated with adulthood and childhood markers of socioeconomic deprivation and to adverse lifestyle factors.</p> <p><b>Methods and Findings:</b> Socioeconomic and lifestyle measures were available in 1040 men and 1298 women from the MIDSPAN Family Study (30–59 years at baseline) together with circulating levels of vitamins A, C, E, and carotenoids (α-carotene, β-carotene, lutein and lycopene). Markers of socioeconomic deprivation in adulthood were consistently as strongly associated with lower vitamin C and carotenoid levels as markers of adverse lifestyle; the inverse association with overcrowding was particularly consistent (vitamin C and carotenoids range from 19.1% [95% CI 30.3–6.0] to 38.8% [49.9–25.3] lower among those in overcrowded residencies). These associations were consistent after adjusting for month, classical CVD risk factors, body mass index, physical activity, vitamin supplements, dietary fat and fibre intake. Similar, but weaker, associations were seen for childhood markers of deprivation. The association of vitamin A or E were strikingly different; several adult adverse lifestyle factors associated with higher levels of vitamin A and E, including high alcohol intake for vitamin A (9.5% [5.7–13.5]) and waist hip ratio for vitamin E (9.5% [4.8–14.4]), with the latter associations partially explained by classical risk factors, particularly cholesterol levels.</p> <p><b>Conclusions:</b> Plasma vitamin C and carotenoids have strong inverse associations with adulthood markers of social deprivation, whereas vitamin A and E appear positively related to specific adverse lifestyle factors. These findings should help researchers better contextualize blood antioxidant vitamin levels by illustrating the potential limitations associated with making causal inferences without consideration of social deprivation.</p&gt