Characterizing molecular and synaptic signatures in mouse models of late-onset Alzheimer's disease independent of amyloid and tau pathology.

INTRODUCTION: MODEL-AD (Model Organism Development and Evaluation for Late-Onset Alzheimer's Disease) is creating and distributing novel mouse models with humanized, clinically relevant genetic risk factors to capture the trajectory and progression of late-onset Alzheimer's disease (LOAD) more accurately. METHODS: We created the LOAD2 model by combining apolipoprotein E4 (APOE4), Trem2*R47H, and humanized amyloid-beta (Aß). Mice were subjected to a control diet or a high-fat/high-sugar diet (LOAD2+HFD). We assessed disease-relevant outcome measures in plasma and brain including neuroinflammation, Aß, neurodegeneration, neuroimaging, and multi-omics. RESULTS: By 18 months, LOAD2+HFD mice exhibited sex-specific neuron loss, elevated insoluble brain Aß42, increased plasma neurofilament light chain (NfL), and altered gene/protein expression related to lipid metabolism and synaptic function. Imaging showed reductions in brain volume and neurovascular uncoupling. Deficits in acquiring touchscreen-based cognitive tasks were observed. DISCUSSION: The comprehensive characterization of LOAD2+HFD mice reveals that this model is important for preclinical studies seeking to understand disease trajectory and progression of LOAD prior to or independent of amyloid plaques and tau tangles. HIGHLIGHTS: By 18 months, unlike control mice (e.g., LOAD2 mice fed a control diet, CD), LOAD2+HFD mice presented subtle but significant loss of neurons in the cortex, elevated levels of insoluble Ab42 in the brain, and increased plasma neurofilament light chain (NfL). Transcriptomics and proteomics showed changes in gene/proteins relating to a variety of disease-relevant processes including lipid metabolism and synaptic function. In vivo imaging revealed an age-dependent reduction in brain region volume (MRI) and neurovascular uncoupling (PET/CT). LOAD2+HFD mice also demonstrated deficits in acquisition of touchscreen-based cognitive tasks.

Renal hyperparathyroidism- a risk factor in the development of encapsulating peritoneal sclerosis.

Background: Encapsulating peritoneal sclerosis (EPS) is a rare complication of prolonged peritoneal dialysis (PD) exposure, characterised by peritoneal thickening, calcification, and fibrosis ultimately presenting with life-threatening bowel obstruction. The presence or role of peritoneal calcification in the pathogenesis of EPS is poorly characterised. We hypothesise that significantly aberrant bone mineral metabolism in patients on PD can cause peritoneal calcification which may trigger the development of EPS. We compared the temporal evolution of bone mineral markers during PD in EPS patients with non-EPS long-term PD controls. Methods: Linear mixed model and logistic regression analysis were used to compare four-monthly serum levels of calcium, phosphate, parathyroid hormone, and alkaline phosphatase (ALP) over the duration of PD exposure in 46 EPS and 46 controls (PD, non-EPS) patients. Results: EPS patients had higher mean calcium (2.51 vs. 2.41 mmol/L) and ALP (248.00 vs. 111.13 IU/L) levels compared with controls (p=0.01 and p<0.001 respectively, maximum likelihood estimation). Logistic regression analysis demonstrated that high serum calcium and phosphate levels during PD were associated with a 4.5 and 2.9 fold increase in the risk of developing EPS respectively. Conclusion: High levels of calcium and phosphate in patients on PD were identified to be risk factors for EPS development. Possible reasons for this may be an imbalance of pro-calcifying factors and calcification inhibitors promoting peritoneal calcification which increases peritoneal stiffness. Mechanical alterations may trigger, unregulated fibrosis and subsequent development of EPS. Improved management of secondary hyperparathyroidism during PD may ultimately diminish the EPS risk.

Characterizing Molecular and Synaptic Signatures in mouse models of Late-Onset Alzheimer's Disease Independent of Amyloid and Tau Pathology.

INTRODUCTION: MODEL-AD is creating and distributing novel mouse models with humanized, clinically relevant genetic risk factors to more accurately mimic LOAD than commonly used transgenic models. METHODS: We created the LOAD2 model by combining APOE4, Trem2*R47H, and humanized amyloid-beta. Mice aged up to 24 months were subjected to either a control diet or a high-fat/high-sugar diet (LOAD2+HFD) from two months of age. We assessed disease-relevant outcomes, including in vivo imaging, biomarkers, multi-omics, neuropathology, and behavior. RESULTS: By 18 months, LOAD2+HFD mice exhibited cortical neuron loss, elevated insoluble brain Aß42, increased plasma NfL, and altered gene/protein expression related to lipid metabolism and synaptic function. In vivo imaging showed age-dependent reductions in brain region volume and neurovascular uncoupling. LOAD2+HFD mice also displayed deficits in acquiring touchscreen-based cognitive tasks. DISCUSSION: Collectively the comprehensive characterization of LOAD2+HFD mice reveal this model as important for preclinical studies that target features of LOAD independent of amyloid and tau.

Monocyte-derived peritoneal macrophages protect C57BL/6 mice against surgery-induced adhesions.

Peritoneal adhesions commonly occur after abdominal or pelvic surgery. These scars join internal organs to each other or to the cavity wall and can present with abdominal or pelvic pain, and bowel obstruction or female infertility. The mechanisms underlying adhesion formation remain unclear and thus, effective treatments are not forthcoming. Peritoneal macrophages accumulate after surgery and previous studies have attributed either pro- or anti-scarring properties to these cells. We propose that there are complex and nuanced responses after surgery with respect to both resident and also monocyte-derived peritoneal macrophage subpopulations. Moreover, we contend that differences in responses of specific macrophage subpopulations in part explain the risk of developing peritoneal scars. We characterized alterations in peritoneal macrophage subpopulations after surgery-induced injury using two strains of mice, BALB/c and C57BL/6, with known differences in macrophage response post-infection. At 14 days post-surgery, BALB/c mice displayed more adhesions compared with C57BL/6 mice. This increase in scarring correlated with a lower influx of monocyte-derived macrophages at day 3 post-surgery. Moreover, BALB/c mice showed distinct macrophage repopulation dynamics after surgery. To confirm a role for monocyte-derived macrophages, we used Ccr2-deficient mice as well as antibody-mediated depletion of CCR2 expressing cells during initial stages of adhesion formation. Both Ccr2-deficient and CCR2-depleted mice showed a significant increase in adhesion formation associated with the loss of peritoneal monocyte influx. These findings revealed an important protective role for monocyte-derived cells in reducing adhesion formation after surgery.

Aspergillus fumigatus-Host Interactions Mediating Airway Wall Remodelling in Asthma.

Asthma is a chronic heterogeneous respiratory condition that is mainly associated with sensitivity to airborne agents such as pollen, dust mite products and fungi. Key pathological features include increased airway inflammation and airway wall remodelling. In particular, goblet cell hyperplasia, combined with excess mucus secretion, impairs clearance of the inhaled foreign material. Furthermore, structural changes such as subepithelial fibrosis and increased smooth muscle hypertrophy collectively contribute to deteriorating airway function and possibility of exacerbations. Current pharmacological therapies focused on airway wall remodelling are limited, and as such, are an area of unmet clinical need. Sensitisation to the fungus, Aspergillus fumigatus, is associated with enhanced asthma severity, bronchiectasis, and hospitalisation. How Aspergillus fumigatus may drive airway structural changes is unclear, although recent evidence points to a central role of the airway epithelium. This review provides an overview of the airway pathology in patients with asthma and fungal sensitisation, summarises proposed airway epithelial cell-fungal interactions and discusses the initiation of a tissue remodelling response. Related findings from in vivo animal models are included given the limited analysis of airway pathology in patients. Lastly, an important role for Aspergillus fumigatus-derived proteases in triggering a cascade of damage-repair events through upregulation of airway epithelial-derived factors is proposed.

The use of mesenchymal stem cells in animal models for gastrointestinal anastomotic leak: A systematic review.

AIM: Anastomotic leak is the most feared complication of gastrointestinal surgery. Mesenchymal stem cell technology is used clinically to promote wound healing; however, the safety and efficacy of this technology on anastomotic healing has yet to be defined. The aim of this study was to investigate whether mesenchymal stem cells confer any benefit when applied to animal models for gastrointestinal anastomotic leak, identify the methodology and how efficacy is assessed. METHODS: The MEDLINE, EMBASE, WebofScience and Cochrane Library databases were interrogated between 1 January1947 to 1 May 2020. All studies where mesenchymal stem cells were applied to laboratory animal leak models to demonstrate a healing effect were considered. All experimental and histological outcomes were examined. Compliance to ARRIVE and current International Consensus was assessed. RESULTS: A total of 1205 studies were screened. Twelve studies reported on 438 gastrointestinal anastomoses in four species using 11 models; seven in the colon. No studies utilised a model with a known leak rate. Significant variance was observed in histological outcomes with efficacy demonstrated in five out of 12 studies. One study demonstrated a benefit in leak rate. Colorectal studies had a greater median ARRIVE compliance, 60.8% (IQR 63.2-64.5) compared to noncolorectal 45.4% (IQR 43.8-49.0). CONCLUSIONS: Mesenchymal stem cell delivery to an animal anastomosis is safe and feasible. Use may confer benefit but findings are currently limited to surrogate histological outcomes. There is consistency in outcome measures reported but variance in how this is assessed. Poor compliance to ARRIVE but good compliance to current international consensus in leak models of the colon was observed.

Ongoing Exposure to Peritoneal Dialysis Fluid Alters Resident Peritoneal Macrophage Phenotype and Activation Propensity.

Peritoneal dialysis (PD) is a more continuous alternative to haemodialysis, for patients with chronic kidney disease, with considerable initial benefits for survival, patient independence and healthcare costs. However, long-term PD is associated with significant pathology, negating the positive effects over haemodialysis. Importantly, peritonitis and activation of macrophages is closely associated with disease progression and treatment failure. However, recent advances in macrophage biology suggest opposite functions for macrophages of different cellular origins. While monocyte-derived macrophages promote disease progression in some models of fibrosis, tissue resident macrophages have rather been associated with protective roles. Thus, we aimed to identify the relative contribution of tissue resident macrophages to PD induced inflammation in mice. Unexpectedly, we found an incremental loss of homeostatic characteristics, anti-inflammatory and efferocytic functionality in peritoneal resident macrophages, accompanied by enhanced inflammatory responses to external stimuli. Moreover, presence of glucose degradation products within the dialysis fluid led to markedly enhanced inflammation and almost complete disappearance of tissue resident cells. Thus, alterations in tissue resident macrophages may render long-term PD patients sensitive to developing peritonitis and consequently fibrosis/sclerosis.

Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent.

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall-epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.

Post-Surgical Peritoneal Scarring and Key Molecular Mechanisms.

Post-surgical adhesions are internal scar tissue and a major health and economic burden. Adhesions affect and involve the peritoneal lining of the abdominal cavity, which consists of a continuous mesothelial covering of the cavity wall and majority of internal organs. Our understanding of the full pathophysiology of adhesion formation is limited by the fact that the mechanisms regulating normal serosal repair and regeneration of the mesothelial layer are still being elucidated. Emerging evidence suggests that mesothelial cells do not simply form a passive barrier but perform a wide range of important regulatory functions including maintaining a healthy peritoneal homeostasis as well as orchestrating events leading to normal repair or pathological outcomes following injury. Here, we summarise recent advances in our understanding of serosal repair and adhesion formation with an emphasis on molecular mechanisms and novel gene expression signatures associated with these processes. We discuss changes in mesothelial biomolecular marker expression during peritoneal development, which may help, in part, to explain findings in adults from lineage tracing studies using experimental adhesion models. Lastly, we highlight examples of where local tissue specialisation may determine a particular response of peritoneal cells to injury.

The Proinflammatory Cytokine IL-36γ Is a Global Discriminator of Harmless Microbes and Invasive Pathogens within Epithelial Tissues.

Epithelial tissues represent vital interfaces between organisms and their environment. As they are constantly exposed to harmful pathogens, innocuous commensals, and environmental microbes, it is essential they sense and elicit appropriate responses toward these different types of microbes. Here, we demonstrate that the epithelial cytokine interleukin-36γ (IL-36γ) acts as a global discriminator of pathogenic and harmless microbes via cell damage and proteolytic activation. We show that intracellular pro-IL-36γ is upregulated by both fungal and bacterial epithelial microbes; yet, it is only liberated from cells, and subsequently processed to its mature, potent, proinflammatory form, by pathogen-mediated cell damage and pathogen-derived proteases. This work demonstrates that IL-36γ senses pathogen-induced cell damage and proteolytic activity and is a key initiator of immune responses and pathological inflammation within epithelial tissues. As an apically located epithelial proinflammatory cytokine, we therefore propose that IL-36γ is critical as the initial discriminator of harmless microbes and invasive pathogens within epithelial tissues.

The Effects of an Acute Bout of Self-Myofascial Release on the Physiological Parameters of Running.

This study examined changes in the physiological parameters of running performance when self-myofascial release (SMR) was used prior to a submaximal run. A total of 16 male recreational runners, between the ages of 27 and 50 years old volunteered for the study. Participants had to complete a running event measuring a 10K or longer in the past 12 months and obtained a V̇O2peak value of 45 mL·kg-l · min-1 to be included in the study. Participants took part in two 40 min treadmill runs at 75% of their V̇O2peak, one session with the use of SMR and the other with 20 min of seated rest prior to the run. Measurements of heart rate, blood lactate concentrations, ventilatory efficiency (V̇E/ V̇O2), RPE, and running velocity were assessed. There was no statistically significant interaction or treatment effect for these variables when SMR was used prior to a 40 min treadmill run (p > .05; heart rate: d = .01, V̇E/ V̇O2: d = .07, RPE: d = .07). Although no positive effects on running performance were found, the lack of negative effects suggests the use of SMR prior to running does not hinder performance.

Regulation of Peritoneal Inflammatory Response to Implant Material Using an Ex Vivo Model System.

BACKGROUND: Implants used in abdominal wall reconstruction are associated with intra-abdominal inflammation that can cause complications such as adhesions, fistulae, or failure of the implant. This study analyzed the inflammatory response of human peritoneum explants when exposed to different implant materials including synthetic and biological (cross-linked and non-cross-linked). MATERIALS AND METHODS: Human peritoneum explants (parietal and visceral) were incubated in culture with implants used for abdominal wall reconstruction. Implants included Permacol (biological implant with chemical cross-linking); Biodesign and Strattice (biological implants without chemical cross-linking); Prolene (synthetic nonabsorbable); and Vicryl (synthetic absorbable). Control peritoneum samples were incubated without implant. Cytokine concentrations and corresponding gene expression were measured by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. Further evaluation included assessment of tissue viability and implant-cytokine adsorption. RESULTS: Incubation of human peritoneal explants with Biodesign or Strattice was associated with a significant reduction in interleukin-6, interleukin-1ß, and tumour necrosis factor alpha protein and gene expression compared with control. These could not be explained by reduced cell viability or implant-cytokine adsorption. Incubation of explants in Biodesign-conditioned media displayed a similar effect to incubation of explants with Biodesign itself. CONCLUSIONS: Human peritoneal explants cultured with different mesh implant materials show an altered inflammatory cytokine response suggesting a tissue-specific response. Downregulation of key inflammatory cytokines by the peritoneum exposed to non-cross-linked biological implants may be mediated by the release of soluble factors from these implants inhibiting cytokine gene expression. This ex vivo human peritoneal system provides a novel preclinical model to investigate peritoneum-implant interactions.

Endothelin-1 mediates Aspergillus fumigatus-induced airway inflammation and remodelling.

BACKGROUND: Asthma is a chronic inflammatory condition of the airways and patients sensitized to airborne fungi such as Aspergillus fumigatus have more severe asthma. Thickening of the bronchial subepithelial layer is a contributing factor to asthma severity for which no current treatment exists. Airway epithelium acts as an initial defence barrier to inhaled spores, orchestrating an inflammatory response and contributing to subepithelial fibrosis. OBJECTIVE: We aimed to analyse the production of pro-fibrogenic factors by airway epithelium in response to A fumigatus, in order to propose novel anti-fibrotic strategies for fungal-induced asthma. METHODS: We assessed the induction of key pro-fibrogenic factors, TGF-ß1, TGF-ß2, periostin and endothelin-1, by human airway epithelial cells and in mice exposed to A fumigatus spores or secreted fungal factors. RESULTS: Aspergillus fumigatus specifically caused production of endothelin-1 by epithelial cells in vitro but not any of the other pro-fibrogenic factors assessed. A fumigatus also induced endothelin-1 in murine lungs, associated with extensive inflammation and airway remodelling. Using a selective endothelin-1 receptor antagonist, we demonstrated for the first time that endothelin-1 drives many features of airway remodelling and inflammation elicited by A fumigatus. CONCLUSION: Our findings are consistent with the hypothesis that elevated endothelin-1 levels contribute to subepithelial thickening and highlight this factor as a possible therapeutic target for difficult-to-treat fungal-induced asthma.

Functional molecules in mesothelial-to-mesenchymal transition revealed by transcriptome analyses.

Peritoneal fibrosis is a common complication of abdominal and pelvic surgery, and can also be triggered by peritoneal dialysis, resulting in treatment failure. In these settings, fibrosis is driven by activated myofibroblasts that are considered to be partly derived by mesothelial-to-mesenchymal transition (MMT). We hypothesized that, if the molecular signature of MMT could be better defined, these insights could be exploited to block this pathological cellular transition. Rat peritoneal mesothelial cells were purified by the use of an antibody against HBME1, a protein present on mesothelial cell microvilli, and streptavidin nanobead technology. After exposure of sorted cells to a well-known mediator of MMT, transforming growth factor (TGF)-ß1, RNA sequencing was undertaken to define the transcriptomes of mesothelial cells before and during early-phase MMT. MMT was associated with dysregulation of transcripts encoding molecules involved in insulin-like growth factor (IGF) and bone morphogenetic protein (BMP) signalling. The application of either recombinant BMP4 or IGF-binding protein 4 (IGFBP4) ameliorated TGF-ß1-induced MMT in culture, as judged from the retention of epithelial morphological and molecular phenotypes, and reduced migration. Furthermore, peritoneal tissue from peritoneal dialysis patients showed less prominent immunostaining than control tissue for IGFBP4 and BMP4 on the peritoneal surface. In a mouse model of TGF-ß1-induced peritoneal thickening, BMP4 immunostaining on the peritoneal surface was attenuated as compared with healthy controls. Finally, genetic lineage tracing of mesothelial cells was used in mice with peritoneal injury. In this model, administration of BMP4 ameliorated the injury-induced shape change and migration of mesothelial cells. Our findings demonstrate a distinctive MMT signature, and highlight the therapeutic potential for BMP4, and possibly IGFBP4, to reduce MMT. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

High-density lipoprotein particle pattern and overall lipid responses to a short-term moderate-intensity aerobic exercise training intervention in patients with chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is associated with abnormal lipid profiles and altered high-density lipoprotein (HDL) particle size patterns. Lower levels of the larger, cardioprotective HDL particles found in CKD may play a role in the increased risk for cardiovascular disease in these patients. The current study was designed to assess the effects of short-term moderate-intensity aerobic exercise training on the HDL particle pattern and overall lipid profiles in stage 3 CKD patients. METHODS: Forty-six men and women with stage 3 CKD were randomized to either exercise (EX, n = 25) or control (CON, n = 21). Those in the EX group completed 16 weeks of supervised moderate-intensity aerobic exercise three times per week. Serum total cholesterol, HDL cholesterol (HDL-C), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), HDL particle size, estimated glomerular filtration rate (eGFR), body composition and peak oxygen uptake (VO2peak) were assessed at baseline and week 16. RESULTS: The rate of compliance in the EX group was 97 ± 7.2%. No change was observed in eGFR over time in either group. There was an 8.2% improvement in VO2peak in the EX group (P = 0.05), while VO2peak decreased in the CON group. HDL-C, TGs, HDL particle size and body composition remained unchanged in both groups. A trend was found for lower total cholesterol (TC) (P = 0.051) and LDL-C (P = 0.07) in the CON group. CONCLUSION: Our findings indicate that a short-term aerobic exercise training intervention in stage 3 CKD patients does not induce changes in HDL particle size or favorable lipid profile modifications.

Mesothelial cells and peritoneal homeostasis.

The mesothelium was traditionally thought to be a simple tissue with the sole function of providing a slippery, nonadhesive, and protective surface to allow easy movement of organs within their body cavities. However, our knowledge of mesothelial cell physiology is rapidly expanding, and the mesothelium is now recognized as a dynamic cellular membrane with many other important functions. When injured, mesothelial cells initiate a cascade of processes leading either to complete regeneration of the mesothelium or the development of pathologies such as adhesions. Normal mesothelial healing is unique in that, unlike with other epithelial-like surfaces, healing appears diffusely across the denuded surface, whereas for epithelium healing occurs solely at the wound edges. This is because of a free-floating population of mesothelial cells which attach to the injured serosa. Taking advantage of this phenomenon, intraperitoneal injections of mesothelial cells have been assessed for their ability to prevent adhesion formation. This review discusses some of the functions of mesothelial cells regarding maintenance of serosal integrity and outlines the mechanisms involved in mesothelial healing. In addition, the pathogenesis of adhesion formation is discussed with particular attention to the potential role of mesothelial cells in both preventing and inducing their development.

Characterization of TLR-induced inflammatory responses in COPD and control lung tissue explants.

PURPOSE: Viruses are a common cause of exacerbations in chronic obstructive pulmonary disease (COPD). They activate toll-like receptors (TLRs) 3, 7, and 8, leading to a pro-inflammatory response. We have characterized the responses of TLR3 and TLR7/8 in lung tissue explants from COPD patients and control smokers. METHODS: We prepared lung whole tissue explants (WTEs) from patients undergoing surgery for confirmed or suspected lung cancer. In order to mimic the conditions of viral infection, we used poly(I:C) for TLR3 stimulation and R848 for TLR7/8 stimulation. These TLR ligands were used alone and in combination. The effects of tumor necrosis factor α (TNFα) neutralization and dexamethasone on TLR responses were examined. Inflammatory cytokine release was measured by enzyme-linked immunosorbent assay and gene expression by quantitative real-time polymerase chain reaction. RESULTS: WTEs from COPD patients released higher levels of pro-inflammatory cytokines compared with WTEs from smokers. Activation of multiple TLRs led to a greater than additive release of TNFα and CCL5. TNFα neutralization and dexamethasone treatment decreased cytokine release. CONCLUSION: This WTE model shows an enhanced response of COPD compared with controls, suggesting an increased response to viral infection. There was amplification of innate immune responses with multiple TLR stimulation.

Mesothelial cells in tissue repair and fibrosis.

Mesothelial cells are fundamental to the maintenance of serosal integrity and homeostasis and play a critical role in normal serosal repair following injury. However, when normal repair mechanisms breakdown, mesothelial cells take on a profibrotic role, secreting inflammatory, and profibrotic mediators, differentiating and migrating into the injured tissues where they contribute to fibrogenesis. The development of new molecular and cell tracking techniques has made it possible to examine the origin of fibrotic cells within damaged tissues and to elucidate the roles they play in inflammation and fibrosis. In addition to secreting proinflammatory mediators and contributing to both coagulation and fibrinolysis, mesothelial cells undergo mesothelial-to-mesenchymal transition, a process analogous to epithelial-to-mesenchymal transition, and become fibrogenic cells. Fibrogenic mesothelial cells have now been identified in tissues where they have not previously been thought to occur, such as within the parenchyma of the fibrotic lung. These findings show a direct role for mesothelial cells in fibrogenesis and open therapeutic strategies to prevent or reverse the fibrotic process.