Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Emphysema.

The activity of PP2A (protein phosphatase 2A), a serine-threonine phosphatase, is reduced by chronic cigarette smoke (SM) exposure and α-1 antitrypsin (AAT) deficiency, and chemical activation of PP2A reduces the loss of lung function in SM-exposed mice. However, the previously studied PP2A-activator tricyclic sulfonamide compound DBK-1154 has low stability to oxidative metabolism, resulting in fast clearance and low systemic exposure. Here we compare the utility of a new more stable PP2A activator, ATUX-792, versus DBK-1154 for the treatment of SM-induced emphysema. ATUX-792 was also tested in human bronchial epithelial cells and a mouse model of AAT deficiency, Serpina1a-e-knockout mice. Human bronchial epithelial cells were treated with ATUX-792 or DBK-1154, and cell viability, PP2A activity, and MAP (mitogen-activated protein) kinase phosphorylation status were examined. Wild-type mice received vehicle, DBK-1154, or ATUX-792 orally in the last 2 months of 4 months of SM exposure, and 8-month-old Serpina1a-e-knockout mice received ATUX-792 daily for 4 months. Forced oscillation and expiratory measurements and histology analysis were performed. Treatment with ATUX-792 or DBK-1154 resulted in PP2A activation, reduced MAP kinase phosphorylation, immune cell infiltration, reduced airspace enlargements, and preserved lung function. Using protein arrays and multiplex assays, PP2A activation was observed to reduce AAT-deficient and SM-induced release of CXCL5, CCL17, and CXCL16 into the airways, which coincided with reduced neutrophil lung infiltration. Our study indicates that suppression of the PP2A activity in two models of emphysema could be restored by next-generation PP2A activators to impact lung function.

LRP1 loss in airway epithelium exacerbates smoke-induced oxidative damage and airway remodeling.

The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor ß activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1-/-) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1-/- mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1-/- mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1-/- mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.

Targeting c-Src Reverses Accelerated GPX-1 mRNA Decay in Chronic Obstructive Pulmonary Disease Airway Epithelial Cells.

Enhanced expression of the cellular antioxidant glutathione peroxidase (GPX)-1 prevents cigarette smoke-induced lung inflammation and tissue destruction. Subjects with chronic obstructive pulmonary disease (COPD), however, have decreased airway GPX-1 levels, rendering them more susceptible to disease onset and progression. The mechanisms that downregulate GPX-1 in the airway epithelium in COPD remain unknown. To ascertain these factors, analyses were conducted using human airway epithelial cells isolated from healthy subjects and human subjects with COPD and lung tissue from control and cigarette smoke-exposed A/J mice. Tyrosine phosphorylation modifies GPX-1 expression and cigarette smoke activates the tyrosine kinase c-Src. Therefore, studies were conducted to evaluate the role of c-Src on GPX-1 levels in COPD. These studies identified accelerated GPX-1 mRNA decay in COPD airway epithelial cells. Targeting the tyrosine kinase c-Src with siRNA inhibited GPX-1 mRNA degradation and restored GPX-1 protein levels in human airway epithelial cells. In contrast, silencing the tyrosine kinase c-Abl, or the transcriptional activator Nrf2, had no effect on GPX-1 mRNA stability. The chemical inhibitors for c-Src (saracatinib and dasanitib) restored GPX-1 mRNA levels and GPX-1 activity in COPD airway cells in vitro. Similarly, saracatinib prevented the loss of lung Gpx-1 expression in response to chronic smoke exposure in vivo. Thus, this study establishes that the decreased GPX-1 expression that occurs in COPD lungs is at least partially due to accelerated mRNA decay. Furthermore, these findings show that targeting c-Src represents a potential therapeutic approach to augment GPX-1 responses and counter smoke-induced lung disease.

Airway Resistance Caused by Sphingomyelin Synthase 2 Insufficiency in Response to Cigarette Smoke.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

Cigarette smoke induction of S100A9 contributes to chronic obstructive pulmonary disease.

S100 calcium-binding protein A9 (S100A9) is elevated in plasma and bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), and aging enhances S100A9 expression in several tissues. Currently, the direct impact of S100A9-mediated signaling on lung function and within the aging lung is unknown. Here, we observed that elevated S100A9 levels in human BALF correlated with age. Elevated lung levels of S100A9 were higher in older mice compared with in young animals and coincided with pulmonary function changes. Both acute and chronic exposure to cigarette smoke enhanced S100A9 levels in age-matched mice. To examine the direct role of S100A9 on the development of COPD, S100a9-/- mice or mice administered paquinimod were exposed to chronic cigarette smoke. S100A9 depletion and inhibition attenuated the loss of lung function, pressure-volume loops, airway inflammation, lung compliance, and forced expiratory volume in 0.05 s/forced vital capacity, compared with age-matched wild-type or vehicle-administered animals. Loss of S100a9 signaling reduced cigarette smoke-induced airspace enlargement, alveolar remodeling, lung destruction, ERK and c-RAF phosphorylation, matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and keratinocyte-derived chemokine (KC) release into the airways. Paquinimod administered to nonsmoked, aged animals reduced age-associated loss of lung function. Since fibroblasts play a major role in the production and maintenance of extracellular matrix in emphysema, primary lung fibroblasts were treated with the ERK inhibitor LY3214996 or the c-RAF inhibitor GW5074, resulting in less S100A9-induced MMP-3, MMP-9, MCP-1, IL-6, and IL-8. Silencing Toll-like receptor 4 (TLR4), receptor for advanced glycation endproducts (RAGE), or extracellular matrix metalloproteinase inducer (EMMPRIN) prevented S100A9-induced phosphorylation of ERK and c-RAF. Our data suggest that S100A9 signaling contributes to the progression of smoke-induced and age-related COPD.

Zika Virus Circulation in Mali.

The circulation of Zika virus (ZIKV) in Mali has not been clearly characterized. Therefore, we conducted a serologic survey of 793 asymptomatic volunteers >15 years of age (2016), and 637 blood donors (2013) to assess the seroprevalence of ZIKV infection in 2 ecoclimatic regions of Mali, tropical savannah and warm semiarid region, using ELISA and seroneutralization assays. The overall seroprevalence was ≈12% and increased with age, with no statistical difference between male and female participants. In the warm semiarid study sites we detected immunological markers of an outbreak that occurred in the late 1990s in 18% (95% CI 13%-23%) of participants. In tropical savannah sites, we estimated a low rate of endemic transmission, with 2.5% (95% CI 2.0%-3.1%) of population infected by ZIKV annually. These data demonstrate the circulation of ZIKV in Mali and provide evidence of a previously unidentified outbreak that occurred in the late 1990s.

Protein Phosphatase 2A Reduces Cigarette Smoke-induced Cathepsin S and Loss of Lung Function.

Rationale: CTSS (cathepsin S) is a cysteine protease that is observed at higher concentrations in BAL fluid and plasma of subjects with chronic obstructive pulmonary disease (COPD). Objectives: To investigate whether CTSS is involved in the pathogenesis of cigarette smoke-induced COPD and determine whether targeting upstream signaling could prevent the disease. Methods: CTSS expression was investigated in animal and human tissue and cell models of COPD. Ctss-/- mice were exposed to long-term cigarette smoke and forced oscillation and expiratory measurements were recorded. Animals were administered chemical modulators of PP2A (protein phosphatase 2A) activity. Measurements and Main Results: Here we observed enhanced CTSS expression and activity in mouse lungs after exposure to cigarette smoke. Ctss-/- mice were resistant to cigarette smoke-induced inflammation, airway hyperresponsiveness, airspace enlargements, and loss of lung function. CTSS expression was negatively regulated by PP2A in human bronchial epithelial cells isolated from healthy nonsmokers and COPD donors and in monocyte-derived macrophages. Modulating PP2A expression or activity, with silencer siRNA or a chemical inhibitor or activator, during acute smoke exposure in mice altered inflammatory responses and CTSS expression and activity in the lung. Enhancement of PP2A activity prevented chronic smoke-induced COPD in mice. Conclusions: Our study indicates that the decrease in PP2A activity that occurs in COPD contributes to elevated CTSS expression in the lungs and results in impaired lung function. Enhancing PP2A activity represents a feasible therapeutic approach to reduce CTSS activity and counter smoke-induced lung disease.

HIV infection model of chronic obstructive pulmonary disease in mice.

Cigarette smoke usage is prevalent in human immunodeficiency virus (HIV)-positive patients, and, despite highly active antiretroviral therapy, these individuals develop an accelerated form of chronic obstructive pulmonary disease (COPD). Studies investigating the mechanisms of COPD development in HIV have been limited by the lack of suitable mouse models. Here we describe a model of HIV-induced COPD in wild-type mice using EcoHIV, a chimeric HIV capable of establishing chronic infection in immunocompetent mice. A/J mice were infected with EcoHIV and subjected to whole body cigarette smoke exposure. EcoHIV was detected in alveolar macrophages of mice. Compared with uninfected mice, concomitant EcoHIV infection significantly reduced forced expiratory flow 50%/forced vital capacity and enhanced distal airspace enlargement following cigarette smoke exposure. Lung IL-6, granulocyte-macrophage colony-stimulating factor, neutrophil elastase, cathepsin G, and matrix metalloproteinase-9 expression was significantly enhanced in smoke-exposed EcoHIV-infected mice. These changes coincided with enhanced IκBα, ERK1/2, p38, and STAT3 phosphorylation and lung cell apoptosis. Thus, the EcoHIV smoke exposure mouse model reproduces several of the pathophysiological features of HIV-related COPD in humans, indicating that this murine model can be used to determine key parameters of HIV-related COPD and to test future therapies for this disorder.

Temporal dynamic of malaria in a suburban area along the Niger River.

BACKGROUND: Even if rainfall and temperature are factors classically associated to malaria, little is known about other meteorological factors, their variability and combinations related to malaria, in association with river height variations. Furthermore, in suburban area, urbanization and growing population density should be assessed in relation to these environmental factors. The aim of this study was to assess the impact of combined environmental, meteorological and hydrological factors on malaria incidence through time in the context of urbanization. METHODS: Population observational data were prospectively collected. Clinical malaria was defined as the presence of parasites in addition to clinical symptoms. Meteorological and hydrological factors were measured daily. For each factors variation indices were estimated. Urbanization was yearly estimated assessing satellite imaging and field investigations. Principal component analysis was used for dimension reduction and factors combination. Lags between malaria incidences and the main components were assessed by cross-correlation functions. Generalized additive model was used to assess relative impact of different environmental components, taking into account lags, and modelling non-linear relationships. Change-point analysis was used to determine transmission periods within years. RESULTS: Malaria incidences were dominated by annual periodicity and varied through time without modification of the dynamic, with no impact of the urbanization. The main meteorological factor associated with malaria was a combination of evaporation, humidity and rainfall, with a lag of 3 months. The relationship between combined temperature factors showed a linear impact until reaching high temperatures limiting malaria incidence, with a lag 3.25 months. Height and variation of the river were related to malaria incidence (respectively 6 week lag and no lag). CONCLUSIONS: The study emphasizes no decreasing trend of malaria incidence despite accurate access to care and control strategies in accordance to international recommendations. Furthermore, no decreasing trend was showed despite the urbanization of the area. Malaria transmission remain increase 3 months after the beginning of the dry season. Addition to evaporation versus humidity/rainfall, nonlinear relationship for temperature and river height and variations have to be taken into account when implementing malaria control programmes.

TLR9 expression is required for the development of cigarette smoke-induced emphysema in mice.

The expression of Toll-like receptor (TLR)-9, a pathogen recognition receptor that recognizes unmethylated CpG sequences in microbial DNA molecules, is linked to the pathogenesis of several lung diseases. TLR9 expression and signaling was investigated in animal and cell models of chronic obstructive pulmonary disease (COPD). We observed enhanced TLR9 expression in mouse lungs following exposure to cigarette smoke. Tlr9(-/-) mice were resistant to cigarette smoke-induced loss of lung function as determined by mean linear intercept, total lung capacity, lung compliance, and tissue elastance analysis. Tlr9 expression also regulated smoke-mediated immune cell recruitment to the lung; apoptosis; expression of granulocyte-colony stimulating factor (G-CSF), the CXCL5 protein, and matrix metalloproteinase-2 (MMP-2); and protein tyrosine phosphatase 1B (PTP1B) activity in the lung. PTP1B, a phosphatase with anti-inflammatory abilities, was identified as binding to TLR9. In vivo delivery of a TLR9 agonist enhanced TLR9 binding to PTP1B, which inactivated PTP1B. Ptp1b(-/-) mice had elevated lung concentrations of G-CSF, CXCL5, and MMP-2, and tissue expression of type-1 interferon following TLR9 agonist administration, compared with wild-type mice. TLR9 responses were further determined in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from nonsmoker, smoker, and COPD donors, and then cultured at air liquid interface. NHBE cells from smokers and patients with COPD expressed more TLR9 and secreted greater levels of G-CSF, IL-6, CXCL5, IL-1ß, and MMP-2 upon TLR9 ligand stimulation compared with cells from nonsmoker donors. Although TLR9 combats infection, our results indicate that TLR9 induction can affect lung function by inactivating PTP1B and upregulating expression of proinflammatory cytokines.

Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner.

BACKGROUND: The use of electronic (e)-cigarettes is increasing rapidly, but their lung health effects are not established. Clinical studies examining the potential long-term impact of e-cigarette use on lung health will take decades. To address this gap in knowledge, this study investigated the effects of exposure to aerosolised nicotine-free and nicotine-containing e-cigarette fluid on mouse lungs and normal human airway epithelial cells. METHODS: Mice were exposed to aerosolised phosphate-buffered saline, nicotine-free or nicotine-containing e-cigarette solution, 1-hour daily for 4 months. Normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface were exposed to e-cigarette vapours or nicotine solutions using a Vitrocell smoke exposure robot. RESULTS: Inhalation of nicotine-containing e-cigarettes increased airway hyper-reactivity, distal airspace enlargement, mucin production, cytokine and protease expression. Exposure to nicotine-free e-cigarettes did not affect these lung parameters. NHBE cells exposed to nicotine-containing e-cigarette vapour showed impaired ciliary beat frequency, airway surface liquid volume, cystic fibrosis transmembrane regulator and ATP-stimulated K+ ion conductance and decreased expression of FOXJ1 and KCNMA1. Exposure of NHBE cells to nicotine for 5 days increased interleukin (IL)-6 and IL-8 secretion. CONCLUSIONS: Exposure to inhaled nicotine-containing e-cigarette fluids triggered effects normally associated with the development of COPD including cytokine expression, airway hyper-reactivity and lung tissue destruction. These effects were nicotine-dependent both in the mouse lung and in human airway cells, suggesting that inhaled nicotine contributes to airway and lung disease in addition to its addictive properties. Thus, these findings highlight the potential dangers of nicotine inhalation during e-cigarette use.

Cathepsin G degradation of phospholipid transfer protein (PLTP) augments pulmonary inflammation.

Phospholipid transfer protein (PLTP) regulates phospholipid transport in the circulation and is highly expressed within the lung epithelium, where it is secreted into the alveolar space. Since PLTP expression is increased in chronic obstructive pulmonary disease (COPD), this study aimed to determine how PLTP affects lung signaling and inflammation. Despite its increased expression, PLTP activity decreased by 80% in COPD bronchoalveolar lavage fluid (BALF) due to serine protease cleavage, primarily by cathepsin G. Likewise, PLTP BALF activity levels decreased by 20 and 40% in smoke-exposed mice and in the media of smoke-treated small airway epithelial (SAE) cells, respectively. To assess how PLTP affected inflammatory responses in a lung injury model, PLTP siRNA or recombinant protein was administered to the lungs of mice prior to LPS challenge. Silencing PLTP at baseline caused a 68% increase in inflammatory cell infiltration, a 120 and 340% increase in ERK and NF-κB activation, and increased MMP-9, IL1ß, and IFN-γ levels after LPS treatment by 39, 140, and 190%, respectively. Conversely, PLTP protein administration countered these effects in this model. Thus, these findings establish a novel anti-inflammatory function of PLTP in the lung and suggest that proteolytic cleavage of PLTP by cathepsin G may enhance the injurious inflammatory responses that occur in COPD.

Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection.

BACKGROUND: Respiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study. METHODS: A549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-ß (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection. RESULTS AND DISCUSSION: RSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-ß). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1ß, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, CXCL1, IP-10/CXCL10, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1ß, IL-5, CXCL12, MIF, LIF, CXCL12 and IFN-γ. Silencing of retinoic acid-inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment. CONCLUSIONS: RSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.

Population genetic structure of Schistosoma bovis and S. curassoni collected from cattle in Mali.

Schistosomiasis is of medical and veterinary importance. Despite the critical situation of schistosomiasis in sub-Saharan Africa, few molecular epidemiological studies have been carried out to determine the role of animals in its transmission. In Mali, it has been over three decades since the last molecular study of animal schistosomes was carried out. It is now urgent to identify circulating strains of the parasite because of potential interactions with other schistosome species, which could complicate disease control. The aim of our work was to study the composition and genetic structure of schistosome populations collected from cattle. The prevalence of schistosome was 23.9%, with the prevalences of Schistosoma bovis (Sb) and S. curassoni (Sc) estimated at 12.6% and 9.8%, respectively. No hybrid strains or S. haematobium were found. The parasites displayed distinct geographical distribution with Sb dominant in Bamako (78.8% and 98% in Central Bamako Slaughterhouse and Sabalibougou Slaughterhouses, respectively) and Sc dominant in Kayes (95.3%). Of the 476 parasites with a complete genetic profile, 60.4% were pure Sc, and were mainly from Kayes. We identified two clusters at the site level (Fst of 0.057 and 0.042 for Sb and Sc, respectively). Cluster 1 was predominantly composed of pure Sb parasites and cluster 2 was mainly composed of pure Sc parasites, from Bamako and Kayes, respectively. Our study shows that cattle schistosomiasis remains endemic in Mali with S. bovis and S. curassoni. A robust genetic structure between the different schistosome populations was identified, which included two clusters based on the geographical distribution of the parasites.

Title: Structure génétique des populations de Schistosoma bovis et S. curassoni collectées chez des bovins au Mali. Abstract: La schistosomiase revêt une grande importance médicale et vétérinaire. Malgré la situation critique de la schistosomiase en Afrique subsaharienne, peu d'études épidémiologiques moléculaires ont été réalisées pour déterminer le rôle des animaux dans sa transmission. Au Mali, cela fait plus de trois décennies que la dernière étude moléculaire des schistosomes animaux a été réalisée. Il est désormais urgent d'identifier les souches circulantes du parasite en raison des interactions potentielles avec d'autres espèces de schistosomes, ce qui pourrait compliquer la lutte contre la maladie. Le but de notre travail était d'étudier la composition et la structure génétique des populations de schistosomes collectées chez des bovins. La prévalence des schistosomes était de 23,9 %, celles de Schistosoma bovis (Sb) et de S. curassoni (Sc) étant respectivement estimées à 12,6 % et 9,8 %. Aucune souche hybride ni S. haematobium n'ont été trouvés. Les parasites présentaient une répartition géographique distincte avec Sb dominant à Bamako (respectivement 78,8 % et 98 % aux Abattoirs Centraux de Bamako et aux Abattoirs de Sabalibougou) et Sc dominant à Kayes (95,3 %). Sur les 476 parasites ayant un profil génétique complet, 60,4 % étaient des Sc purs, et provenaient principalement de Kayes. Nous avons identifié deux clusters au niveau du site (Fst de 0,057 et 0,042 pour Sb et Sc, respectivement). Le groupe 1 était principalement composé de parasites Sb purs et le groupe 2 était principalement composé de parasites Sc purs, provenant respectivement de Bamako et de Kayes. Notre étude montre que la schistosomiase bovine reste endémique au Mali, avec S. bovis and S. curassoni. Une structure génétique robuste entre les différentes populations de schistosomes a été identifiée, comprenant deux groupes basés sur la répartition géographique des parasites.

Human-water interactions associated to cercarial emergence pattern and their influences on urinary schistosomiasis transmission in two endemic areas in Mali.

BACKGROUND: Mali is known to be a schistosomiasis-endemic country with a limited supply of clean water. This has forced many communities to rely on open freshwater bodies for many human-water contact (HWC) activities. However, the relationship between contact with these water systems and the level of schistosome infection is currently receiving limited attention. This study assessed human-water interactions including cercarial emergence pattern and their influences on urinary schistosomiasis transmission in two communities in the Kayes district of Mali. METHODS: We carried out a parasitological study first in children in September 2021, then a cross-sectional study of quantitative observations of human-water contact activities in the population, and finally a study of snail infectivity at contact points in September 2022. The study took place in two communities, Fangouné Bamanan and Diakalèl in the Kayes region of western Mali. The chronobiological study focused on cercarial release from naturally infected snails. Released cercariae were molecularly genotyped by targeting the cox1 region, and the ITS and 18S ribosmal DNA gene (18S rDNA) regions of the DNA. Links between sociodemographic parameters, human water-contact points and hematuria were established using multivariate statistical analysis or the logistic regression model. RESULTS: The main factor predisposing the 97 participants to water contact was domestic activity (62.9%). Of the 378 snails collected at 14 sampling sites, 27 (7.1%) excreted schistosome cercariae, with 15.0% (19/126) at Fangouné Bamanan and 3.3% (8/252) at Diakalel. The release of Schistosoma cercariae shows three different patterns in Fangouné Bamanan: (i) an early release peak (6:00-8:00 AM), (ii) a mid-day release peak (10:00 AM-12:00 PM) and (iii) a double peak: (6:00-8:00 AM) and (6:00-8:00 PM) cercariae release; and two release patterns in Diakalel: early release (6:00-8:00 AM) and (ii) mid-day release (12:00-2:00 PM). All cercariae released during early diurnal (6:00-8:00 AM) or nocturnal emission patterns (6:00-8:00 PM) were hybrids parasite having an cox1 S. bovis or S. curassoni associated with an ITS and 18S rDNA of S. haematobium while the cercariae released during diurnal, or mid-day patterns (8:00 AM-6:00 PM) were pure S. haematobium. CONCLUSIONS: Our study showed that domestic activity is the main source of exposure in the Kayes region. Two and three cercariae emission patterns were observed at Diakalel and Fangouné Bamanan respectively. These results suggest that the parasite adapts to the human-water contact period in order to increase its infectivity.

A duplex tetra-primer ARMS-PCR assay to discriminate three species of the Schistosoma haematobium group: Schistosoma curassoni, S. bovis, S. haematobium and their hybrids.

BACKGROUND: The use of applications involving single nucleotide polymorphisms (SNPs) has greatly increased since the beginning of the 2000s, with the number of associated techniques expanding rapidly in the field of molecular research. Tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR) is one such technique involving SNP genotyping. It has the advantage of amplifying multiple alleles in a single reaction with the inclusion of an internal molecular control. We report here the development of a rapid, reliable and cost-effective duplex T-ARMS-PCR assay to distinguish between three Schistosoma species, namely Schistosoma haematobium (human parasite), Schistosoma bovis and Schistosoma curassoni (animal parasites), and their hybrids. This technique will facilitate studies of population genetics and the evolution of introgression events. METHODS: During the development of the technique we focused on one of the five inter-species internal transcribed spacer (ITS) SNPs and one of the inter-species 18S SNPs which, when combined, discriminate between all three Schistosoma species and their hybrid forms. We designed T-ARMS-PCR primers to amplify amplicons of specific lengths for each species, which in turn can then be visualized on an electrophoresis gel. This was further tested using laboratory and field-collected adult worms and field-collected larval stages (miracidia) from Spain, Egypt, Mali, Senegal and Ivory Coast. The combined duplex T-ARMS-PCR and ITS + 18S primer set was then used to differentiate the three species in a single reaction. RESULTS: The T-ARMS-PCR assay was able to detect DNA from both species being analysed at the maximum and minimum levels in the DNA ratios (95/5) tested. The duplex T-ARMS-PCR assay was also able to detect all hybrids tested and was validated by sequencing the ITS and the 18S amplicons of 148 of the field samples included in the study. CONCLUSIONS: The duplex tetra-primer ARMS-PCR assay described here can be applied to differentiate between Schistosoma species and their hybrid forms that infect humans and animals, thereby providing a method to investigate the epidemiology of these species in endemic areas. The addition of several markers in a single reaction saves considerable time and is of long-standing interest for investigating genetic populations.

Ultrasound aspects and risk factors associated with urogenital schistosomiasis among primary school children in Mali.

BACKGROUND: Urogenital schistosomiasis is endemic in Mali and is a major cause of serious morbidity in large parts of the world. This disease is responsible for many socio-economic and public health issues. The aim of this study was to investigate the impact of the disease on morbidity and to describe demographic and socioeconomic factors in relation to the status of children with urogenital schistosomiasis in Mali. METHODS: We conducted a cross-sectional study in November 2021 of 971 children aged 6 to 14 years selected at random from six schools in three districts in the Kayes Region of Mali. Demographic and socioeconomic data were collected on survey forms. Clinical data were collected following a medical consultation. Hematuria was systematically searched for through the use of strips. The search for Schistosoma haematobium eggs in urine was done via the filtration method. The urinary tract was examined by ultrasound. Associations between each of these variables and disease infection were tested using multivariate logistic regression. RESULTS: The overall prevalence of urinary schistosomiasis detected was 50.2%. The average intensity of infection was 36 eggs/10 ml of urine. The associated risk factors for urogenital schistosomiasis showed that children who bathed, used the river/pond as a domestic water source, and who habitually urinated in the river/pond were more affected (P < 0.05). Children with farming parents were most affected (P = 0.032). The collection of clinical signs revealed that boys had more pollakiuria (58.6%) and dysuria (46.4%) than girls. Ultrasound data showed that focal lesion rates were recorded in all villages with the lowest rate in Diakalel (56.1%). Ultrasound and parasitological findings showed that irregularity and thickening were strongly associated with urinary schistosomiasis (P < 0.0001). CONCLUSIONS: Schistosoma haematobium infection was still endemic in the study site despite more than a decade of mass treatment with praziquantel. However, the high percentage of symptoms associated with high intensity reinforces the idea that further studies in terms of schistosomiasis-related morbidity are still needed.

Genetic profiles of Schistosoma haematobium parasites from Malian transmission hotspot areas.

BACKGROUND: Although schistosomiasis is a public health issue in Mali, little is known about the parasite genetic profile. The purpose of this study was to analyze the genetic profile of the schistosomes of Schistosoma haematobium group in school-aged children in various sites in Mali. METHODS: Urine samples were collected from 7 to 21 November 2021 and subjected to a filtration method for the presence S. haematobium eggs. The study took place in two schistosomiasis endemic villages (Fangouné Bamanan and Diakalèl), qualified as hotspots according to the World Health Organization (WHO) definition. Molecular genotyping on both Cox1 and ITS2/18S was used for eggs' taxonomic assignation. RESULTS: A total of 970 miracidia were individually collected from 63 school-aged children and stored on Whatman FTA cards for molecular analysis. After genotyping 42.0% (353/840) and 58.0% (487/840) of miracidia revealed Schistosoma bovis and S. haematobium Cox1 profiles, respectively; 95.7 (885/925) and 4.3% (40/925) revealed S. haematobium and S. haematobium/S. curassoni profiles for ITS/18S genes, respectively. There was a significant difference in the Cox1 and ITS2/18S profile distribution according to the village (P < 0.0001). Overall, 45.6% (360/789) were hybrids, of which 72.0% (322/447) were from Diakalèl. Three hybrids' profiles (Sb/Sc_ShxSc with 2.3%; Sb/Sc_ShxSh with 40.5%; Sh_ShxSc with 2.8%) and one pure profile (Sh_ShxSh with 54.4%) were identified. CONCLUSION: Our findings show, for the first time to our knowledge, high prevalence of hybrid schistosomes in Mali. More studies are needed on population genetics of schistosomes at the human and animal interface to evaluate the parasite's gene flow and its consequences on epidemiology of the disease as well as the transmission to humans.

Small-Molecule Activation of Protein Phosphatase 2A Counters Bleomycin-Induced Fibrosis in Mice.

The activity of protein phosphatase 2A (PP2A), a serine-threonine phosphatase, is reduced in the lung fibroblasts of idiopathic pulmonary fibrosis (IPF) patients. The objective of this study was to determine whether the reactivation of PP2A could reduce fibrosis and preserve the pulmonary function in a bleomycin (BLM) mouse model. Here, we present a new class of direct small-molecule PP2A activators, diarylmethyl-pyran-sulfonamide, exemplified by ATUX-1215. ATUX-1215 has improved metabolic stability and bioavailability compared to our previously described PP2A activators. Primary human lung fibroblasts were exposed to ATUX-1215 and an older generation PP2A activator in combination with TGFß. ATUX-1215 treatment enhanced the PP2A activity, reduced the phosphorylation of ERK and JNK, and reduced the TGFß-induced expression of ACTA2, FN1, COL1A1, and COL3A1. C57BL/6J mice were administered 5 mg/kg ATUX-1215 daily following intratracheal instillation of BLM. Three weeks later, forced oscillation and expiratory measurements were performed using the Scireq Flexivent System. ATUX-1215 prevented BLM-induced lung physiology changes, including the preservation of normal PV loop, compliance, tissue elastance, and forced vital capacity. PP2A activity was enhanced with ATUX-1215 and reduced collagen deposition within the lungs. ATUX-1215 also prevented the BLM induction of Acta2, Ccn2, and Fn1 gene expression. Treatment with ATUX-1215 reduced the phosphorylation of ERK, p38, JNK, and Akt and the secretion of IL-12p70, GM-CSF, and IL1α in BLM-treated animals. Delayed treatment with ATUX-1215 was also observed to slow the progression of lung fibrosis. In conclusion, our study indicates that the decrease in PP2A activity, which occurs in fibroblasts from the lungs of IPF subjects, could be restored with ATUX-1215 administration as an antifibrotic agent.

TLR4 protein contributes to cigarette smoke-induced matrix metalloproteinase-1 (MMP-1) expression in chronic obstructive pulmonary disease.

Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease and alters expression of proteolytic enzymes that contribute to disease pathology. Previously, we reported that smoke exposure leads to the induction of matrix metalloproteinase-1 (MMP-1) through the activation of ERK1/2, which is critical to the development of emphysema. To date, the upstream signaling pathway by which cigarette smoke induces MMP-1 expression has been undefined. This study demonstrates that cigarette smoke mediates MMP-1 expression via activation of the TLR4 signaling cascade. In vitro cell culture studies demonstrated that cigarette smoke-induced MMP-1 was regulated by TLR4 via MyD88/IRAK1. Blockade of TLR4 or inhibition of IRAK1 prevented cigarette smoke induction of MMP-1. Mice exposed to acute levels of cigarette smoke exhibited increased TLR4 expression. To further confirm the in vivo relevance of this signaling pathway, rabbits exposed to acute cigarette smoke were found to have elevated TLR4 signaling and subsequent MMP-1 expression. Additionally, lungs from smokers exhibited elevated TLR4 and MMP-1 levels. Therefore, our data indicate that TLR4 signaling, through MyD88 and IRAK1, plays a predominant role in MMP-1 induction by cigarette smoke. The identification of the TLR4 pathway as a regulator of smoke-induced protease production presents a series of novel targets for future therapy in chronic obstructive pulmonary disease.