The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo.

Trueperella pyogenes can cause severe pulmonary disease in swine, but the mechanism of pathogenesis is not well defined. T. pyogenes-induced damage to porcine bronchial epithelial cells (PBECs), porcine precision-cut lung slices (PCLS), and respiratory epithelium of mice remains unknown. In this study, we used T. pyogenes 20121 to infect PBECs in air-liquid interface conditions and porcine PCLS. T. pyogenes could adhere to, colonize, and induce cytotoxic effect on PBECs and the luminal surface of bronchi in PCLS, which damaged the bronchiolar epithelium. Moreover, bronchiolar epithelial cells showed extensive degeneration in the lungs of infected mice. Furthermore, western blot showed that the NOD-like receptor (NLR)/C-terminal caspase recruitment domain (ASC)/caspase-1 axis and nuclear factor-kappa B pathway were involved in inflammation in PCLS and lungs of mice, which also confirms that porcine PCLS provide a platform to analyze the pulmonary immune response. Meanwhile, the levels of p-c-Jun N-terminal kinase, p-extracellular signal-regulated kinase, and p-protein kinase B (AKT) were increased significantly, which indicated the mitogen-activated protein kinase and Akt pathways were also involved in inflammation in T. pyogenes-infected mice. In addition, we used T. pyogenes 20121 to infect tumor necrosis factor-alpha (tnf-α-/-) mice, and the results indicated that apoptosis and injury in respiratory epithelium of infected tnf-α-/- mice were alleviated. Thus, the pro-inflammatory cytokine TNF-α played a role in apoptosis and the respiratory epithelium injury in mouse lungs. Collectively, our study provides insight into the inflammatory injury induced by T. pyogenes and suggests that blocking NLR may be a potential therapeutic strategy against T. pyogenes infection.

Menthol flavoring in e-cigarette condensate causes pulmonary dysfunction and cytotoxicity in precision cut lung slices.

E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent menthol-induced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease.

N-acetyl cysteine mitigates lung damage and inflammation after chlorine exposure in vivo and ex vivo.

The objective of this study was to explore the effects of antioxidant treatments, specifically N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA), in a mouse model of chlorine (Cl2)-induced lung injury. Additionally, the study aimed to investigate the utility of pig precision-cut lung slices (PCLS) as an ex vivo alternative for studying the short-term effects of Cl2 exposure and evaluating antioxidant treatments. The toxicological responses were analyzed in Cl2-exposed mice (inflammation, airway hyperresponsiveness (AHR)) and PCLS (viability, cytotoxicity, inflammatory mediators). Airways contractions were assessed using a small ventilator for mice and electric-field stimulation (EFS) for PCLS. Antioxidant treatments were administered to evaluate their effects. In Cl2-exposed mice, NAC treatment did not alleviate AHR, but it did reduce the number of neutrophils in bronchoalveolar lavage fluid and inflammatory mediators in lung tissue. In PCLS, exposure to Cl2 resulted in concentration-dependent toxicity, impairing the lung tissue's ability to respond to EFS-stimulation. NAC treatment increased viability, mitigated the toxic responses caused by Cl2 exposure, and maintained contractility comparable to unexposed controls. Interestingly, NACA did not provide any additional treatment effect beyond NAC in both models. In conclusion, the establishment of a pig model for Cl2-induced lung damage supports further investigation of NAC as a potential treatment. However, the lack of protective effects on AHR after NAC treatment in mice suggests that NAC alone may not be sufficient as a complete treatment for Cl2 injuries. Optimization of existing medications with a polypharmacy approach may be more successful in addressing the complex sequelae of Cl2-induced lung injury.

Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways.

INTRODUCTION: The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease. METHODS: After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function. RESULTS: Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1-/-) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol. CONCLUSION: Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

Electronic Cigarette Exposure Increases the Severity of Influenza a Virus Infection via TRAIL Dysregulation in Human Precision-Cut Lung Slices.

The use of electronic nicotine dispensing systems (ENDS), also known as electronic cigarettes (ECs), is common among adolescents and young adults with limited knowledge about the detrimental effects on lung health such as respiratory viral infections and underlying mechanisms. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a protein of the TNF family involved in cell apoptosis, is upregulated in COPD patients and during influenza A virus (IAV) infections, but its role in viral infection during EC exposures remains unclear. This study was aimed to investigate the effect of ECs on viral infection and TRAIL release in a human lung precision-cut lung slices (PCLS) model, and the role of TRAIL in regulating IAV infection. PCLS prepared from lungs of nonsmoker healthy human donors were exposed to EC juice (E-juice) and IAV for up to 3 days during which viral load, TRAIL, lactate dehydrogenase (LDH), and TNF-α in the tissue and supernatants were determined. TRAIL neutralizing antibody and recombinant TRAIL were utilized to determine the contribution of TRAIL to viral infection during EC exposures. E-juice increased viral load, TRAIL, TNF-α release and cytotoxicity in IAV-infected PCLS. TRAIL neutralizing antibody increased tissue viral load but reduced viral release into supernatants. Conversely, recombinant TRAIL decreased tissue viral load but increased viral release into supernatants. Further, recombinant TRAIL enhanced the expression of interferon-ß and interferon-λ induced by E-juice exposure in IAV-infected PCLS. Our results suggest that EC exposure in human distal lungs amplifies viral infection and TRAIL release, and that TRAIL may serve as a mechanism to regulate viral infection. Appropriate levels of TRAIL may be important to control IAV infection in EC users.

Functional changes in long-term incubated rat precision-cut lung slices.

BACKGROUND: Respiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days. METHODS: Rat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation. RESULTS: Cultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin. CONCLUSIONS: Overall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

Organophosphorus pesticides exhibit compound specific effects in rat precision-cut lung slices (PCLS): mechanisms involved in airway response, cytotoxicity, inflammatory activation and antioxidative defense.

Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is the predominant toxic mechanism, OP may induce pneumonia and formation of lung edema after poisoning and during clinical treatment as life-threatening complication. To investigate the underlying mechanisms, rat precision-cut lung slices (PCLS) were exposed to the OP parathion, malathion and their biotransformation products paraoxon and malaoxon (100-2000 µmol/L). Airway response, metabolic activity, release of LDH, cytokine expression and oxidative stress response were analyzed. A concentration-dependent inhibition of airway relaxation was observed after exposure with the oxon but not with the thion-OP. In contrast, cytotoxic effects were observed for both forms in higher concentrations. Increased cytokine expression was observed after exposure to parathion and paraoxon (IL-6, GM-CSF, MIP-1α) and IL-6 expression was dependent on NFκB activation. Intracellular GSH levels were significantly reduced by all four tested OP but an increase in GSSG and HO-1 expression was predominantly observed after malaoxon exposure. Pretreatment with the antioxidant N-acetylcysteine reduced malaoxon but not paraoxon-induced cytotoxicity. PCLS as a 3D lung model system revealed OP-induced effects depending on the particular OP. The experimental data of this study contribute to a better understanding of OP toxicity on cellular targets and may be a possible explanation for the variety of clinical outcomes induced by different OP.

Genetic profiles of different ethnicities living in Karachi as regards to tobacco-metabolising enzyme systems and the risk of oral cancer.

OBJECTIVE: To detect polymorphisms of CYP1A1, GSTM1 and GSTT1 gene loci among various tobacco-consuming ethnicities in an urban centre, and to relate these with susceptibility to oral cancer. METHODS: The cross-sectional, case-control study was conducted at Ziauddin University, Karachi, and the Dow University of Health Sciences, Karachi, from 2011 to 2016, and comprised patients having oral squamous cell carcinoma in group A, with oral precancerous lesions in group B, and tobacco habit-matched controls in group C. Routine histopathology was followed by molecular analysis through polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism techniques. Data was analysed using SPSS 20. RESULTS: Of the 358 subjects, 150(42%) were in group A, 100(28%) in group B, and 108(30%) in group C. There were 190(53.1%) Urdu-speaking subjects, 42(11.7%) Memoni-speaking, 37(10.3%) Sindhi-speaking, 34(9.5%) Balochi-speaking, 25(7%) Pashto-speaking, 15(4.2%) Punjabi-speaking, and 15(4.2) of other ethnicities. Among the Urdu-speaking ethnicity, CYP1A1 MspI heterozygous variant was the most prevalent genotype ingroup A 50(66.7%), group B 37(62.7%) and group C 36(64.3%). The homozygous variant was equally distributed in group A 8(13.5%) and group B 10(13.3%), while it remained quite low in group C 4(7.1%). Homozygous genotype was most common in Pashto-speaking subjects in group A 4(57.1%). In Urdu-speaking subjects, GSTM1-null genotype was mostly found in group B 19(32.2%), while GSTT1-null genotype was most common in group A 12(16%). Other than Urdu-speaking, GSTM1-null variant was most frequent in Sindhi-speaking subjects in group B 8(80%). CONCLUSIONS: Intra-ethnic distribution of tobacco-metabolising enzyme genes can be considered an important contributor to oral cancer risk in the population of Karachi.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Best Practices and Progress in Precision-Cut Liver Slice Cultures.

Thirty-five years ago, precision-cut liver slices (PCLS) were described as a promising tool and were expected to become the standard in vitro model to study liver disease as they tick off all characteristics of a good in vitro model. In contrast to most in vitro models, PCLS retain the complex 3D liver structures found in vivo, including cell-cell and cell-matrix interactions, and therefore should constitute the most reliable tool to model and to investigate pathways underlying chronic liver disease in vitro. Nevertheless, the biggest disadvantage of the model is the initiation of a procedure-induced fibrotic response. In this review, we describe the parameters and potential of PCLS cultures and discuss whether the initially described limitations and pitfalls have been overcome. We summarize the latest advances in PCLS research and critically evaluate PCLS use and progress since its invention in 1985.

Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Prevalence and determinants of post-traumatic stress disorder among road traffic accident survivors: a prospective survey at selected hospitals in southern Ethiopia.

BACKGROUND: Post-traumatic stress disorder (PTSD) is prevalent among road traffic accident survivors (RTA), yet the psychological welfare of the persons has largely been ignored as health care professionals focus more on managing physical injuries. Many literatures from other parts of the world have addressed the issue of post-traumatic stress disorder among road traffic accident survivors, but such studies are mostly unavailable in sub-Saharan Africa, especially in Ethiopia. Therefore, this study examined the prevalence and determinants of PTSD among RTA survivors attending selected hospitals in southern Ethiopia. METHODS: Institution based cross-sectional study design was employed from April 1/2018-Sep 30/2019. Data were collected from a sample of consecutively selected 423 RTA survivors through an interviewer-administered technique. A pre-tested post-traumatic stress disorder Checklist-Specific version (PCL-S) tool was used to screen PTSD. Data were entered and analysed using SPSS 22 software. A logistic regression model was fitted to identify determinants of PTSD. An adjusted odds ratio (AOR) with a 95% confidence interval was computed to determine the level of significance with a p-value of less than 0.05. RESULT: A total of 416 participants were included in the study and the response rate was 98.6%. The prevalence of probable PTSD among RTA survivors was 15.4% (64). After adjusting for the effects of potential confounding variables; time since accident (30-90 days) (AOR = 0.33; 95%CI (0.15, 0.73), history of previous road traffic accident (AOR = 2.67; 95%CI (1.23, 5.77), depressive symptoms (AOR = 2.5, 95% CI (1.10, 6.10)) and common mental disorder (AOR = 12.78, 95% CI (5.56, 29.36)) were significant determinants of PTSD. CONCLUSION: The prevalence of probable PTSD in the current study was high (15.4%). Time since accident, history of a previous road traffic accident, having depressive symptoms and common mental disorder were significant determinants of PTSD. RTA survivors attending adult Emergency and orthopedic clinics need to be screened for PTSD and get appropriate management.

The gut microbiota metabolite indole alleviates liver inflammation in mice.

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p-cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision-cut liver slices prepared from control mice, Kupffer cell (KC)-depleted mice, and obese mice ( ob/ ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose-dependently reduced the LPS-induced up-regulation of proinflammatory mediators at both mRNA and protein levels in precision-cut liver slices prepared from control or ob/ ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF-κB pathway and downstream proinflammatory gene up-regulation. Indole also prevented LPS-induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4ß-hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.-Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.-B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice.

Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. METHODS: We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. RESULTS: Low µM concentrations of Nintedanib (1 µM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. CONCLUSIONS: Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.

Store-operated calcium entry is required for sustained contraction and Ca2+ oscillations of airway smooth muscle.

KEY POINTS: Airway hyper-responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (ASMCs). Agonist-induced Ca2+ oscillations underlie this contraction of ASMCs and the magnitude of this contraction is proportional to the Ca2+ oscillation frequency. Sustained contraction and Ca2+ oscillations require an influx of extracellular Ca2+ , although the mechanisms and pathways mediating this Ca2+ influx during agonist-induced ASMC contraction are not well defined. By inhibiting store-operated calcium entry (SOCE) or voltage-gated Ca2+ channels (VGCCs), we show that SOCE, rather than Ca2+ influx via VGCCs, provides the major Ca2+ entry pathway into ASMCs to sustain ASMCs contraction and Ca2+ oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper-responsiveness in asthma. ABSTRACT: Asthma is characterized by airway hyper-responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca2+ oscillations within airway smooth muscle cells (ASMCs). Sustained Ca2+ oscillations require a Ca2+ influx to replenish Ca2+ losses across the plasma membrane. Our previous studies implied store-operated calcium entry (SOCE) as the major pathway for this Ca2+ influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975A and GSK5498A) as well as L-type voltage-gated Ca2+ channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca2+ oscillations and SOCE-mediated Ca2+ influx in ASMCs within mouse precision-cut lung slices. We found that both GSK7975A and GSK5498A were able to fully relax methacholine-induced airway contraction by abolishing the Ca2+ oscillations, in a manner similar to that observed in zero extracellular Ca2+ ([Ca2+ ]e ). In addition, GSK7975A and GSK5498A inhibited increases in intracellular Ca2+ ([Ca2+ ]i ) in ASMCs with depleted Ca2+ -stores in response to increased [Ca2+ ]e , demonstrating a response consistent with the inhibition of SOCE. However, GSK7975A and GSK5498A did not reduce Ca2+ release via IP3 receptors stimulated with IP3 released from caged-IP3 . By contrast, nifedipine and nimodipine only partially reduced airway contraction, Ca2+ oscillation frequency and SOCE-mediated Ca2+ influx. These data suggest that SOCE is the major Ca2+ influx pathway for ASMCs with respect to sustaining agonist-induced airway contraction and the underlying Ca2+ oscillations. The mechanisms of SOCE may therefore form novel targets for new bronchodilators.

[Correlation between specific and nonspecific posttraumatic stress disorder symptoms with healthcare consumption among 340 French soldiers]. / État de stress post-traumatique et consommation de soins sur l'année écoulée: étude menée auprès de 340 militaires français de cinq unités combattantes de l'armée de terre.

BACKGROUND: The psychotraumatic disorders are often difficult to diagnose because the specific symptoms of posttraumatic stress disorder (revival, hyperarousal, avoidance) are rarely a direct demand for health care: for reasons determined by the psychopathological structure of trauma, its symptomatology and course, the psychotraumatised subjects seek a care system for nonspecific psychological or somatoform symptoms: depressive episode, cognitive disorders, other anxiety disorders, histrionic and obsessive symptoms, changes in personality, pain disorders and somatization. Somatic pain may also result from a war injury and psychosomatic complications, addictive or consequences of risk behaviours during the evolution of posttraumatic stress disorder. OBJECTIVES: To establish a correlation between the PCLS and the evaluation of the healthcare consumption in a military population. METHODS: We conducted a multicenter epidemiological study analyzing the PCLS and a questionnaire assessing health care consumption. The PCLS has been studied in various forms: quantitative (17 to 85), in qualitative classes (<33, 33 to 43 and ≥44), and in five sub-dimensions (flashbacks, avoidance, dissociation, depression and hyperactivity). The sub-dimension revival was then studied item by item. The criteria used care consumption over the last twelve months is the numbers of days of sick leave, days of unavailability (of certain jobs or military activities) and consultations. RESULTS: Our population of 340 subjects cannot be considered representative of the French military population even if only a few characteristics differ. Sixteen of 340 subjects show a positive PCLS is 4.70% of our sample. PCLS average of 23 (±9.4) with a median of 19 objectifying much of PCLS have almost zero score. Validating our main hypothesis, we found a statistically significant relationship between elements of the PCLS and variables care consumption: this link exists mainly between the score, classes and sub-dimensions of the PCLS in one hand and number of days of sick leave and unavailability on the other hand. DISCUSSION: Towards a strategy for tracking psychotraumatic disorders, could be developed a score of health care consumption which would include the number of days of sick leave and unavailability, the number and quality of medical consultations, the number and quality of drug and laboratory requirements, the number of hospitalisations. To the identification of posttraumatic stress disorder, the PCLS score as well as the consumer healthcare score are valuable tools but do not replace the subjectivity of the clinical relationship: return to this shared subjectivity with the practitioner remains a diagnostic dimension, but also therapeutic, fundamental.

Primary cutaneous lymphomas: A clinical and histological study of 99 cases in Isfahan, Iran.

BACKGROUND: Primary cutaneous lymphomas (PCLs) represent a heterogeneous group of T- and B-cell lymphomas that present in the skin with no evidence of extracutaneous disease at the time of diagnosis. The aim of this study was to assess and report the epidemiological characteristics of PCLs in Isfahan, Isfahan Province, Iran - as a main province of Iran. MATERIALS AND METHODS: A total of 99 patients were recruited over a recent 10-year period (2003-2013) with diagnosis of PCLs; the patients were classified according to the The World Health Organization/European Organization for Research and Treatment of Cancer (WHO-EORTC) criteria. Mean and standard deviations (SDs) were used to describe continuous data, numbers, and percentages for categorical data. Statistical significance was defined as P < 0.05. RESULTS: The patients comprised 45 men and 54 women aged 5-80 years (median 36) at diagnosis. The male-to-female ratio was 1:1.2. Histological examination showed features of primary cutaneous B-cell lymphomas (PCBCLs) in four cases. The mean ± SD age in primary cutaneous T-cell lymphomas (PCTCLs) and PCBCLs was 37.9 ± 16.5 years and 39.7 ± 9.1 years, respectively (P = 0.72). The mean ± SD latent period between the time of diagnosis and initiation of skin lesions in men and women was 2.3 ± 4.1 years and 5.9 ± 10.1 years, respectively (P = 0.02). The most frequent subtypes were mycosis fungoides (MFs) (86.9%) followed by Sιzary syndrome (SS) (4%). Five patients died from PCL-related deaths. CONCLUSION: The distinguishing epidemiologic characteristics of PCL in Iran are the absence of a male predominance and a lower age of diagnosis. The study highlights the ethnic or regional variations in the clinicoepidemiological characteristics of PCLs.

Precision-cut lung slices as an ex vivo model to study Pneumocystis murina survival and antimicrobial susceptibility.

IMPORTANCE: Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

AChE reactivation in precision-cut lung slices following organophosphorus compound poisoning.

Precision-cut lung slices (PCLS) are a suitable model for analyzing the acetylcholinesterase (AChE) activity and subsequent effects after exposure to organophosphorus (OP) compounds. In this study, the AChE activity was determined in intact PCLS for the first time. Since the current standard therapy for OP poisoning (atropine + oxime + benzodiazepine) lacks efficiency, reliable models to study novel therapeutic substances are needed. Models should depict pathophysiological mechanisms and help to evaluate the beneficial effects of new therapeutics. Here PCLS were exposed to three organophosphorus nerve agents (OPNAs): sarin (GB), cyclosarin (GF), and VX. They were then treated with three reactivators: HI-6, obidoxime (OBI), and a non-oxime (NOX-6). The endpoints investigated in this study were the AChE activity and the airway area (AA) change. OPNA exposure led to very low residual AChE activities. Depending on the reactivator properties different AChE reactivation results were measured. GB-inhibited PCLS-AChE was reactivated best, followed by VX and GF. To substantiate these findings and to understand the connection between the molecular and the functional levels in a more profound way the results were correlated to the AA changes. These investigations underline the importance of reactivator use and point to the possibilities for future improvements in the treatment of OPNA-exposed victims.