[Large scale teaching in pathology]. / Pédagogie à grande échelle en ACP.

Medical education is currently facing great changes that affect all medical specialties, including anatomical pathology. Due to rapidly increasing medical knowledge and diagnostic complexity, we are living an era of teaching resources mutualization. We present different tools that allow large numbers of students to access courses, self-evaluations, and competencies assessments. MOOC platforms and e-learning platforms are central to these new online tools, which include the French National Platform of Medical Specialties, dedicated to the teaching of 50,000 medical residents in France. We also discuss "serious games" and the use of images and virtual slides in anatomical pathology teaching. These new modalities can deliver essential knowledge to large student populations, but they must be used in conjunction with adapted teacher-led courses focusing on competencies and professional skills in order to be fully effective.

The Efficacy of MAG-DHA for Correcting AA/DHA Imbalance of Cystic Fibrosis Patients.

Omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementations are thought to improve essential fatty acid deficiency (EFAD) as well as reduce inflammation in Cystic Fibrosis (CF), but their effectiveness in clinical studies remains unknown. The aim of the study was to determine how the medical food containing docosahexaenoic acid monoglyceride (MAG-DHA) influenced erythrocyte fatty acid profiles and the expression levels of inflammatory circulating mediators. We conducted a randomized, double blind, pilot trial including fifteen outpatients with Cystic Fibrosis, ages 18⁻48. The patients were divided into 2 groups and received MAG-DHA or a placebo (sunflower oil) for 60 days. Patients took 8 × 625 mg MAG-DHA softgels or 8 × 625 mg placebo softgels every day at bedtime for 60 days. Lipid analyses revealed that MAG-DHA increased docosahexaenoic acid (DHA) levels and decrease arachidonic acid (AA) ratio (AA/DHA) in erythrocytes of CF patients following 1 month of daily supplementation. Data also revealed a reduction in plasma human leukocyte elastase (pHLE) complexes and interleukin-6 (IL-6) expression levels in blood samples of MAG-DHA supplemented CF patients. This pilot study indicates that MAG-DHA supplementation corrects erythrocyte AA/DHA imbalance and may exert anti-inflammatory properties through the reduction of pHLE complexes and IL6 in blood samples of CF patients. TRIAL REGISTRATION: Pro-resolving Effect of MAG-DHA in Cystic Fibrosis (PREMDIC), NCT02518672.

Potential Application of Eicosapentaenoic Acid Monoacylglyceride in the Management of Colorectal Cancer.

BACKGROUND: There is increasing evidence that marine omega-3 oils are involved in the reduction of cancer risk and progression. However, the anticancer effect of omega-3 monoglyceride on colorectal cancer has yet to be assessed. The goal of this study was to evaluate the anti-cancer effects of eicosapentaenoic acid monoglyceride (MAG-EPA) in HCT116 colorectal carcinoma cells. METHODS: The effect of MAG-EPA was evaluated in vitro on HCT116 cells and in vivo on mouse model of HCT116 xenograft. RESULTS: Our data reveal that MAG-EPA decreased cell proliferation and induced apoptosis in HCT116 cells. In a xenograft mouse model, daily per os administration of MAG-EPA reduced tumor growth. Furthermore, MAG-EPA treatments decreased EGFR, VEGFR, and AKT activation pathways and reduced VEGF and HIF1α expression levels in tumors. CONCLUSION: MAG-EPA may promote apoptosis and inhibit growth of tumors by suppressing EGFR and VEGFR activation pathways. Altogether, these data provide new evidence regarding the mode of action of MAG-EPA in colorectal cancer cells.

MAG-EPA reduces severity of DSS-induced colitis in rats.

Ulcerative colitis (UC) is a chronic disease characterized by diffuse inflammation of the intestinal mucosa of the large bowel. Omega-3 (ω3) fatty acid supplementation has been associated with a decreased production of inflammatory cytokines involved in UC pathogenesis. The aim of this study was to determine the preventive and therapeutic potential of eicosapentaenoic acid monoglyceride (MAG-EPA) in an in vivo rats model of UC induced by dextran sulfate sodium (DSS). DSS rats were untreated or treated per os with MAG-EPA. Morphological, histological, and biochemical analyses were performed following MAG-EPA administrations. Morphological and histological analyses revealed that MAG-EPA pretreatment (12 days pre-DSS) and treatment (6 days post-DSS) exhibited strong activity in reducing severity of disease in DSS rats. Following MAG-EPA administrations, tissue levels of the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 were markedly lower compared with rats treated only with DSS. MAG-EPA per os administration decrease neutrophil infiltration in colon tissues, as depicted by myelohyperoxidase activity. Results also revealed a reduced activation of NF-κB pathways correlated with a decreased expression of COX-2 in colon homogenates derived from MAG-EPA-pretreated and treated rats. Tension measurements performed on colon tissues revealed that contractile responses to methacholine and relaxing effect induced by sodium nitroprusside were largely increased following MAG-EPA treatment. The combined treatment of MAG-EPA and vitamin E displayed an antagonistic effect on anti-inflammatory properties of MAG-EPA in DSS rats.

Proresolving Action of Docosahexaenoic Acid Monoglyceride in Lung Inflammatory Models Related to Cystic Fibrosis.

Cystic fibrosis (CF) is a hereditary, chronic disease of the exocrine glands, characterized by the production of viscid mucus that obstructs the pancreatic ducts and bronchi, leading to infection and fibrosis. ω3 fatty acid supplementations are known to improve the essential fatty acid deficiency as well as reduce inflammation in CF. The objective of this study was to determine the effects of docosahexaenoic acid monoacylglyceride (MAG-DHA) on mucin overproduction and resolution of airway inflammation in two in vitro models related to CF. Isolated human bronchi reverse permeabilized with CF transmembrane conductance regulator (CFTR) silencing (si) RNA and stable Calu3 cells expressing a short hairpin (sh) RNA directed against CFTR (shCFTR) were used. Lipid analyses revealed that MAG-DHA increased DHA/arachidonic acid (AA) ratio in shCFTR Calu-3 cells. MAG-DHA treatments, moreover, resulted in a decreased activation of Pseudomonas aeruginosa LPS-induced NF-κB in CF and non-CF Calu-3 cells. Data also revealed a reduction in MUC5AC, IL-6, and IL-8 expression levels in MAG-DHA-treated shCFTR cells stimulated, or not, with LPS. Antiinflammatory properties of MAG-DHA were also investigated in a reverse-permeabilized human bronchi model with CFTR siRNA. After MAG-DHA treatments, messenger RNA transcript levels for MUC5AC, IL-6, and IL-8 were markedly reduced in LPS-treated CFTR siRNA bronchi. MAG-DHA displays antiinflammatory properties and reduces mucin overexpression in Calu-3 cells and human bronchi untreated or treated with P. aeruginosa LPS, a finding consistent with the effects of resolvinD1, a known antiinflammatory mediator.

Resolvin E1 normalizes contractility, Ca2+ sensitivity and smooth muscle cell migration rate in TNF-α- and IL-6-pretreated human pulmonary arteries.

Pulmonary hypertension (PH) is a rare disease in which pathophysiology is characterized by an increase in proinflammatory mediators, chronic endothelial dysfunctions, and a high migration rate of smooth muscle cells (SMC). Over the course of the last decade, various treatments have been proposed to relax the pulmonary arteries, none of which have been effective in resolving PH. Our hypothesis is that artery-relaxing drugs are not the long-term solution, but rather the inhibition of tissue inflammation, which underlies human pulmonary artery (HPA) dysfunctions that lead to abnormal vasoconstriction. The goal of the present study was to assess the anti-inflammatory effects of resolvin E1 (RvE1) with concomitant effects on SMC migration and on HPA reactivity. The role and mode of action of RvE1 and its precursor, monoacylglyceride eicosapentaenoic acid were assessed on HPA under proinflammatory conditions, involving a combined pretreatment with 10 ng/ml TNF-α and 10 ng/ml IL-6. Our results show that TNF-α and IL-6 treatment induced hyperreactivity and Ca(2+) hypersensitivity in response to pharmaco-mechanical stimuli, including 80 mM KCl, 1 µM phorbol 12-13-dibutyrate, and 30 nM U-46619. Furthermore, the proinflammatory treatment increased the migration rate of SMC isolated from HPA. The phosphorylation level of regulatory contractile proteins (CPI-17, MYPT-1), and proinflammatory signaling pathways (c-Fos, c-Jun, NF-κB) were also significantly increased compared with control conditions. Conversely, 300 nM RvE1 was able to normalize all of the above abnormal events triggered by proinflammation. In conclusion, RvE1 can resolve human arterial hyperreactivity via the resolution of inflammatory markers.

Effect of docosahexaenoic acid monoacylglyceride on systemic hypertension and cardiovascular dysfunction.

ω-3 Fatty acid supplementation has been associated with lower blood pressure. Cardiovascular diseases are also known to be linked directly to an increase in ω-6 and a reduction in ω-3 fatty acid levels in blood circulation and tissues. To determine the effect of docosahexaenoic acid monoglycerides (MAG-DHA) on blood pressure, lipid profiles, and vascular remodeling in rats fed a high-fat/high-carbohydrate (HFHC) diet. Studies were performed in male rats subjected to 8 wk of HFHC diet supplemented or not with 3 g/day MAG-DHA. After 8 wk of daily MAG-DHA treatment, rats in the HFHC + MAG-DHA group had lower arterial blood pressure and heart rate compared with the HFHC group. Moreover, MAG-DHA prevented the increase aortic wall thickness, whereas lipid analysis of aortic tissues revealed an increase in DHA/AA ratio correlated with the production of resolvin D2 and D3 metabolites. Histological analysis revealed that MAG-DHA prevented the development of LVH in the HFHC group. Serum lipid profile analysis further showed a decrease in total cholesterol (TC) and LDL, including very low-density lipoprotein (VLDL) and triglyceride (TG) levels, together with an increase in HDL levels after 8 wk of MAG-DHA treatment compared with the HFHC group. Furthermore, daily MAG-DHA treatment resulted in reduced proinflammatory marker levels such as CRP, IL-6, TNFα, and IL-1ß. Altogether, these findings revealed that per os administration of MAG-DHA prevents HFHC-diet induced hypertension and LVH in rats.

Reversal of IL-13-induced inflammation and Ca(2+) sensitivity by resolvin and MAG-DHA in association with ASA in human bronchi.

The aim of this study was to investigate the effects of resolvin D1 (RvD1), as well as the combined treatment of docosahexaenoic acid monoglyceride (MAG-DHA) and acetylsalicylic acid (ASA), on the resolution of inflammation markers and Ca(2+) sensitivity in IL-13-pretreated human bronchi (HB). Tension measurements performed with 300 nM RvD1 largely abolished (50%) the over-reactivity triggered by 10 ng/ml IL-13 pretreatment and reversed hyper Ca(2+) sensitivity. Addition of 300 nM 17(S)-HpDoHE, the metabolic intermediate between DHA and RvD1, displayed similar effects. In the presence of 100 µM ASA (a COX inhibitor), the inhibitory effect of 1 µM MAG-DHA on muscarinic tone was further amplified, but not in the presence of Ibuprofen. Western blot analysis revealed that the combined treatment of MAG-DHA and ASA upregulated GPR-32 expression and downregulated cytosolic TNFα detection, hence preventing IκBα degradation and p65-NFκB phosphorylation. The Ca(2+) sensitivity of HB was also quantified on ß-escin permeabilized preparations. The presence of ASA potentiated the inhibitory effects of MAG-DHA in reducing the Ca(2+) hypersensitivity triggered by IL-13 by decreasing the phosphorylation levels of the PKC-potentiated inhibitor protein-17 regulatory protein (CPI-17). In summary, MAG-DHA combined with ASA, as well as exogenously added RvD1, may represent valuable assets against critical AHR disorder.

Docosapentaenoic acid monoacylglyceride reduces inflammation and vascular remodeling in experimental pulmonary hypertension.

n-3 Polyunsaturated fatty acids (n-3 PUFA) have been shown to reduce inflammation and proliferation of pulmonary artery smooth muscle cells under pathophysiological conditions. However, the anti-inflammatory effect of the newly synthesized docosapentaenoic acid monoacylglyceride (MAG-DPA) on key signaling pathways in pulmonary hypertension (PH) pathogenesis has yet to be assessed. The aim of the present study was to determine the effects of MAG-DPA on pulmonary inflammation and remodeling occurring in a rat model of PH, induced by a single injection of monocrotaline (MCT: 60 mg/kg). Our results demonstrate that MAG-DPA treatment for 3 wk following MCT injection resulted in a significant improvement of right ventricular hypertrophy (RVH) and a reduction in Fulton's Index (FI). Morphometric analyses revealed that the wall thickness of pulmonary arterioles was significantly lower in MCT + MAG-DPA-treated rats compared with controls. This result was further correlated with a decrease in Ki-67 immunostaining. Following MAG-DPA treatments, lipid analysis showed a consistent increase in DPA together with lower levels of arachidonic acid (AA), as measured in blood and tissue samples. Furthermore, in MCT-treated rats, oral administration of MAG-DPA decreased NF-κB and p38 MAPK activation, leading to a reduction in MMP-2, MMP-9, and VEGF expression levels in lung tissue homogenates. Altogether, these data provide new evidence regarding the mode of action of MAG-DPA in the prevention of pulmonary hypertension induced by MCT.

Resolvin D1 reverses reactivity and Ca2+ sensitivity induced by ET-1, TNF-α, and IL-6 in the human pulmonary artery.

Pulmonary hypertension (PH) is a rare and progressive disease characterized by an inflammatory status and vessel wall remodeling, resulting in increased pulmonary artery resistance. During the last decade, treatments have been proposed; most of them target the endothelial pathways that stimulate smooth muscle cell relaxation. However, PH remains associated with significant morbidity. We hypothesized that inflammation plays a crucial role in the severity of the abnormal vasoconstriction in PH. The goal of this study was to assess the effects of resolvin D1 (RvD1), a potent anti-inflammatory agent, on the pharmacological reactivity of human pulmonary arteries (HPAs) via an in vitro model of induced hyperreactivity. The effects of RvD1 and monoacylglyceride compounds were measured on contractile activity and Ca(2+) sensitivity developed by HPAs that had been pretreated (or not) under proinflammatory conditions with either 10 ng/ml TNF-α or 10 ng/ml IL-6 or under hyperreactive conditions with 5 nM endothelin-1. The results demonstrated that, compared with controls, 24-h pretreatment with TNF-α, IL-6, or endothelin-1 increased reactivity and Ca(2+) sensitivity of HPAs as revealed by agonist challenges with 80 mM KCl, 1 µM serotonin (5-hydroxytryptamine), 30 nM U-46619, and 1 µM phorbol 12,13-dibutyrate. However, 300 nM RvD1 as well as 1 µM monoacylglyceride-docosapentaenoic acid monoglyceride strongly reversed the overresponsiveness induced by both proinflammatory and hyperreactive treatments. In pretreated pulmonary artery smooth muscle cells, Western blot analyses revealed that RvD1 treatment decreased the phosphorylation level of CPI-17 and expression of transmembrane protein member 16A while increasing the detection of G protein-coupled receptor 32. The present data demonstrate that RvD1, a trihydroxylated docosahexaenoic acid derivative, decreases induced overreactivity in HPAs via a reduction in CPI-17 phosphorylation and transmembrane protein member 16A expression.

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties.

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.

Dashboards to Support Implementation of the Quebec Alzheimer Plan: Evaluation Study With Regional and Professional Considerations.

BACKGROUND: Health organizations face the critical task of executing and overseeing comprehensive health care. To address the challenges associated with this task, evidence-based dashboards have emerged as valuable tools. Since 2016, the regional health organizations of Quebec, Canada, have been responsible for ensuring implementation of the Quebec Alzheimer Plan (QAP), a provincial plan that aims to reinforce the capacity of primary care services to detect, diagnose, and treat persons with dementia. Despite the provincial scope of the QAP, the diverse material and human resources across regions introduce variability in the interest, utility, and specific needs associated with these dashboards. OBJECTIVE: The aim of this study was to assess the interest and utility of dashboards to support the QAP implementation, as well as to determine the needs for improving these aspects according to the perspectives of various types of professionals involved across regions. METHODS: An evaluative study using qualitative methods was conducted within a collaborative research approach involving different stakeholders, including the ministerial advisor and the four project managers responsible for supporting the implementation of the QAP, as well as researchers/scientific advisors. To support these organizations, we developed tailored, 2-page paper dashboards, detailing quantitative data on the prevalence of dementia, the use of health services by persons with dementia, and achievements and challenges of the QAP implementation in each organization's jurisdiction. We then conducted 23 focus groups with the managers and leading clinicians involved in the implementation of the QAP of each regional health organization. Real-time notes were taken using a structured observation grid. Content analysis was conducted according to different regions (organizations with university mandates or nearby organizations, labeled "university/peripheral"; organizations for which only part of the territory is in rural areas, labeled "mixed"; and organizations in remote or isolated areas, labeled "remote/isolated") and according to different types of participants (managers, leading clinicians, and other participants). RESULTS: Participants from organizations in all regions expressed interest in these dashboards and found them useful in several ways. However, they highlighted the need for indicators on orphan patients and other health care providers. Differences between regions were observed, particularly in the interest in continuity of care in university/peripheral regions and the need for diagnostic tools adapted to the culture in remote/isolated regions. CONCLUSIONS: These dashboards support the implementation of an Alzheimer Plan and contribute to the emergence of a learning health care system culture. This project allows each region to increase its monitoring capacity for the implementation of the QAP and facilitates reflection among individuals locally carrying out the implementation. The perspectives expressed will guide the preparation of the next iteration of the dashboards.

A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker.

Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on ∼1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I-IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.

Transfer of Mexiletine into Breast Milk: A Case Report.

Background: Mexiletine is a class 1B antiarrhythmic agent, used to treat ventricular arrhythmias, and noncardiac-related problems such as myotonia. Limited safety data are available on the transfer of this drug into breast milk. Case Report: We report the case of a woman diagnosed with myotonia congenita who breastfed two children after two consecutive pregnancies. During the breastfeeding of the first and the second infant, she collected, respectively, five and seven samples at 0, 2, 4, 6, and 8 hours and 0, 1, 2, 3, 4, 6, and 8 hours after taking 200 mg of mexiletine thrice daily for seven doses. One week after the collection, samples were analyzed with a validated liquid chromatography tandem mass spectrometry method. No side effect was observed in either child according to the mother. Results: Using the mean milk concentrations, it is estimated that an exclusively breastfed infant would receive a maximum of 5.14% of the initial pediatric mexiletine dosage. We calculated a maximum of 2.67% for the first infant in our case, considering a nonexclusive breastfeeding. Maximal concentrations were observed 1-2 hours after the dose of mexiletine. Results seem to be in accordance with the two cases previously published. Conclusions: Mexiletine transfers into breast milk in low levels. However, results are obtained from only one woman. Therefore, caution should be used when mexiletine is prescribed to breastfeeding women. More cases are needed to evaluate the interindividual variability and to guide women regarding breastfeeding with mexiletine.

The Three W's of Acetaminophen In Children: Who, Why, and Which Administration Mode.

Acetaminophen is one of the oldest medications commonly administered in children. Its efficacy in treating fever and pain is well accepted among clinicians. However, the available evidence supporting the use of acetaminophen's different modes of administration remains relatively scarce and poorly known. This short report summarizes the available evidence and provides a framework to guide clinicians regarding a rational use of acetaminophen in children.

Bronchial inflammation induced PKCζ over-expression: involvement in mechanical properties of airway smooth muscle.

Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.

Centralizing neurofibromatosis experimental tool knowledge with the NF Research Tools Database.

Experimental tools and resources, such as animal models, cell lines, antibodies, genetic reagents and biobanks, are key ingredients in biomedical research. Investigators face multiple challenges when trying to understand the availability, applicability and accessibility of these tools. A major challenge is keeping up with current information about the numerous tools available for a particular research problem. A variety of disease-agnostic projects such as the Mouse Genome Informatics database and the Resource Identification Initiative curate a number of types of research tools. Here, we describe our efforts to build upon these resources to develop a disease-specific research tool resource for the neurofibromatosis (NF) research community. This resource, the NF Research Tools Database, is an open-access database that enables the exploration and discovery of information about NF type 1-relevant animal models, cell lines, antibodies, genetic reagents and biobanks. Users can search and explore tools, obtain detailed information about each tool as well as read and contribute their observations about the performance, reliability and characteristics of tools in the database. NF researchers will be able to use the NF Research Tools Database to promote, discover, share, reuse and characterize research tools, with the goal of advancing NF research. Database URL: https://tools.nf.synapse.org/.

Docosahexaenoic acid derivative prevents inflammation and hyperreactivity in lung: implication of PKC-Potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kD in asthma.

The effects of a newly synthesized docosahexaenoic acid (DHA) derivative, CRBM-0244, on lung inflammation and airway hyperresponsiveness were determined in an in vitro model of TNF-α-stimulated human bronchi and in an in vivo model of allergic asthma. Mechanical tension measurements revealed that CRBM-0244 prevented bronchial hyperresponsiveness in TNF-α-pretreated human bronchi. Moreover, treatment with CRBM-0244 resulted in a decrease in NF-κB activation and cyclooxygenase-2 (COX-2) overexpression triggered by TNF-α. The inhibition of peroxisome proliferator-activated receptor-γ with GW9662 abolished the CRBM-0244-mediated anti-inflammatory effects. CRBM-0244 reduced the Ca(2+) sensitivity of bronchial smooth muscle through a decrease in the phosphorylation and expression of the PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17). Results also revealed an overexpression of CPI-17 protein in lung biopsies derived from patients with asthma. Furthermore, the presence of specialized enzymes such as 5-lipoxygenase and 15-lipoxygenase in the lung may convert CRBM-0244 into active mediators, leading to the resolution of inflammation. The in vivo anti-inflammatory properties of CRBM-0244 were also investigated in a guinea pig model of allergic asthma. After oral administration of CRBM-0244, airway leukocyte recruitment, airway mucus, ovalbumin-specific IgE, and proinflammatory markers such as TNF-α and COX-2 were markedly reduced. Hence, CRBM-0244 treatment prevents airway hyperresponsiveness, Ca(2+) hypersensitivity, and the overexpression of CPI-17 in lung tissue. Together, these findings provide key evidence regarding the mode of action of CRBM-0244 in the lung, and point to new therapeutic strategies for modulating inflammation in patients with asthma.

NCS 613, a potent and specific PDE4 inhibitor, displays anti-inflammatory effects on human lung tissues.

Chronic inflammation is a hallmark of pulmonary diseases, which leads to lung parenchyma destruction (emphysema) and obstructive bronchiolitis occurring in both chronic obstructive pulmonary disease and asthma. Inflammation is strongly correlated with low intracellular cAMP levels and increase in specific cAMP hydrolyzing activity. The aim of the present study was to investigate the role of the cyclic phosphodiesterase type 4 (PDE4) in human lung and to determine the effects of NCS 613, a new PDE4 inhibitor, on lung inflammation and bronchial hyperresponsiveness. High cAMP-PDE activities were found in the cytosoluble fractions from human lung parenchyma and distal bronchi. PDE4 (rolipram sensitive) represented 40% and 56% of total cAMP-PDE activities in the above-corresponding tissues. Moreover, PDE4A, PDE4B, PDE4C, and PDE4D isoforms were detected in all three subcellular fractions (cytosolic, microsomal, and nuclear) with differential distributions according to specific variants. Pharmacological treatments with NCS 613 significantly decreased PDE4 activity and reduced IκBα degradation in cultured parenchyma, both of which are usually correlated with a lower inflammation status. Moreover, NCS 613 pretreatment potentiated isoproterenol-induced relaxations in human distal bronchi, while reducing TNF-α-induced hyperresponsiveness in cultured bronchi, as assessed in the presence of methacholine, U-46619, or histamine. This reducing effect of NCS 613 on human bronchi hyperresponsiveness triggered by TNF-α was related to a lower expression level of PDE4B and PDE4C, as well as a downregulation of the phosphorylated forms of p38-MAPK, CPI-17, and MYPT-1, which are known to control tone. In conclusion, specific PDE4 inhibitors, such as NCS 613, may represent an alternative and isoform-specific approach toward reducing human lung inflammation and airway overreactivity.