Cigarette smoke alters inflammatory genes and the extracellular matrix - investigations on viable sections of peripheral human lungs.

Chronic obstructive pulmonary disease (COPD) is a complex chronic respiratory disorder often caused by cigarette smoke. Cigarette smoke contains hundreds of toxic substances. In our study, we wanted to identify initial mechanisms of cigarette smoke induced changes in the distal lung. Viable slices of human lungs were exposed 24 h to cigarette smoke condensate, and the dose-response profile was analyzed. Non-toxic condensate concentrations and lipopolysaccharide were used for further experiments. COPD-related protein and gene expression was measured. Cigarette smoke condensate did not induce pro-inflammatory cytokines and most inflammation-associated genes. In contrast, lipopolysaccharide significantly induced IL-1α, IL-1ß, TNF-α and IL-8 (proteins) and IL1B, IL6, and TNF (genes). Interestingly, cigarette smoke condensate induced metabolism- and extracellular matrix-associated proteins and genes, which were not influenced by lipopolysaccharide. Also, a significant regulation of CYP1A1 and CYP1B1, as well as MMP9 and MMP9/TIMP1 ratio, was observed which resembles typical findings in COPD. In conclusion, our data show that cigarette smoke and lipopolysaccharide induce significant responses in human lung tissue ex vivo, giving first hints that COPD starts early in smoking history.

Rhinovirus-induced Human Lung Tissue Responses Mimic Chronic Obstructive Pulmonary Disease and Asthma Gene Signatures.

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection.

INTRODUCTION: Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%-40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. METHODS: It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl-) channel modulators to investigate their role during the HRSV life-cycle. RESULTS: We demonstrate the requirement for TMEM16A, a calcium-activated Cl- channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. DISCUSSION: These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.

Human lung tissue provides highly relevant data about efficacy of new anti-asthmatic drugs.

Subgroups of patients with severe asthma are insensitive to inhaled corticosteroids and require novel therapies on top of standard medical care. IL-13 is considered one of the key cytokines in the asthma pathogenesis, however, the effect of IL-13 was mostly studied in rodents. This study aimed to assess IL-13 effect in human lung tissue for the development of targeted therapy approaches such as inhibition of soluble IL-13 or its receptor IL-4Rα subunit. Precision-cut lung slices (PCLS) were prepared from lungs of rodents, non-human primates (NHP) and humans. Direct effect of IL-13 on human lung tissue was observed on inflammation, induction of mucin5AC, and airway constriction induced by methacholine and visualized by videomicroscopy. Anti-inflammatory treatment was evaluated by co-incubation of IL-13 with increasing concentrations of IL-13/IL-13 receptor inhibitors. IL-13 induced a two-fold increase in mucin5AC secretion in human bronchial tissue. Additionally, IL-13 induced release of proinflammatory cytokines eotaxin-3 and TARC in human PCLS. Anti-inflammatory treatment with four different inhibitors acting either on the IL-13 ligand itself (anti-IL-13 antibody, similar to Lebrikizumab) or the IL-4Rα chain of the IL-13/IL-4 receptor complex (anti-IL-4Rα #1, similar to AMG 317, and #2, similar to REGN668) and #3 PRS-060 (a novel anticalin directed against this receptor) could significantly attenuate IL-13 induced inflammation. Contrary to this, IL-13 did not induce airway hyperresponsiveness (AHR) in human and NHP PCLS, although it was effective in rodent PCLS. Overall, this study demonstrates that IL-13 stimulation induces production of mucus and biomarkers of allergic inflammation in human lung tissue ex-vivo but no airway hyperresponsiveness. The results of this study show a more distinct efficacy than known from animals models and a clear discrepancy in AHR induction. Moreover, it allows a translational approach in inhibitor profiling in human lung tissue.

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), and interleukin 1 alpha (IL-1α) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

Coagulation factor XII regulates inflammatory responses in human lungs.

Increased procoagulant activity in the alveolar compartment and uncontrolled inflammation are hallmarks of the acute respiratory distress syndrome (ARDS). Here, we investigated whether the contact phase system of coagulation is activated and may regulate inflammatory responses in human lungs. Components of the contact phase system were characterized in bronchoalveolar lavage fluids (BALF) from 54 ARDS patients and 43 controls, and their impact on cytokine/chemokine expression in human precision cut lung slices (PCLS) was assessed by a PCR array. Activation of the contact system, associated with high levels of coagulation factor XIIa (Hageman factor, FXIIa), plasma kallikrein and bradykinin, occurred rapidly in ARDS lungs after the onset of the disease and virtually normalized within one week from time of diagnosis. FXII levels in BALF were higher in ARDS non-survivors than survivors and were positively correlated with tumor necrosis factor (TNF)-α concentration. FXII induced the production and release of interleukin (IL)-8, IL-1ß, IL-6, leukemia inhibitory factor (LIF), CXCL5 and TNF-α in human PCLS in a kallikrein-kinin-independent manner. In conclusion, accumulation of FXII in ARDS lungs may contribute to the release of pro-inflammatory mediators and is associated with clinical outcome. FXII inhibition may thus offer a novel and promising therapeutic approach to antagonize overwhelming inflammatory responses in ARDS lungs without interfering with vital haemostasis.

Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality.

BACKGROUND: Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days. METHODS: PCLS were cultured for 15 days and various parameters were assessed at different time points. RESULTS: Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC501d = 4 × 10-8 M vs. EC5015d = 4 × 10-6 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture. CONCLUSIONS: Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays.

FXII promotes proteolytic processing of the LRP1 ectodomain.

BACKGROUND: Factor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described. METHODS: The effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively. RESULTS: Here, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media. CONCLUSIONS: FXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain. GENERAL SIGNIFICANCE: FXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.

Functional testing of an inhalable nanoparticle based influenza vaccine using a human precision cut lung slice technique.

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10(3) µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1ß, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing.

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1ß) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1ß. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1ß levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.

Aortopexy in infants and children--long-term follow-up in twenty patients.

BACKGROUND: Aortopexy has become an established surgical procedure for the treatment of tracheomalacia (TM) in infants and children. The aim of the present study was to evaluate the clinical outcome and respiratory function after aortopexy in the long term. METHODS: Between 1992 and 2006, 20 patients (6 female, 14 male) with TM were treated by bronchoscopically monitored pexis of the aorta via a right anterior thoracotomy. Patient age ranged from 4 months to 11 years (mean: 29 months). Five infants had previous surgery of esophageal atresia or tracheo-esophageal fistulae, and five other patients were operated on for gastroesophageal reflux. Postoperative tidal expiratory flow (TEF25%) was compared to age-related values. RESULTS: Mean follow-up was 7.8 years (range: 13 months to 10.7 years). There was no early or late mortality. Most patients (n = 16) showed immediate and permanent relief of symptoms. Compared to corresponding age groups, median TEF25% was slightly but not significantly decreased after aortopexy (p = 0.15). In one patient a re-aortopexy was necessary. Another patient experienced recurrent tracheo-esophageal fistula 3 years after aortopexy. CONCLUSIONS: The bronchoscopically guided aortopexy is an efficient and simple method in the surgical treatment of TM in infants and children. The follow-up data in this series of 20 patients showed improvement of respiratory function and permanent relief of symptoms in the long term.

Disseminated Aspergillus fumigatus infection with consecutive mitral valve endocarditis in a lung transplant recipient.

Aspergillus infection is a known complication of lung transplantation and remains associated with high mortality rates. The manifestation of the infection varies from simple colonization of the lung to disseminated complicated infections. Early Aspergillus infection has been rarely observed in a small number of lung transplant recipients; most cases occur during the late post-operative period. The pulmonary involvement has often been described as the first clinical localization of the disease. Although other various forms of Aspergillus infection are not uncommonly encountered after lung transplantation, Aspergillus mitral valve endocarditis is rare. We present a case of disseminated Aspergillus fumigatus infection with consecutive mitral valve endocarditis having developed 78 days after double-lung transplantation for cystic fibrosis.

Surgical versus endovascular treatment of acute thoracic aortic rupture: a single-center experience.

BACKGROUND: Surgical management of acute thoracic aortic ruptures is controversial, especially in patients with preexisting comorbidities; associated mortality and paraplegia rates remain high. It was our objective to evaluate whether treating these patients acutely with endovascular stent grafts would improve their outcome. METHODS: From November 1999 to February 2002 a total of 54 patients, age 28 to 83 years, were admitted to our institution with an acute rupture of the thoracic aorta (24 ruptured aneurysms, 14 perforated type B dissections, 16 traumatic ruptures). Twenty-eight patients were managed surgically using cardiopulmonary bypass (group 1), and 26 patients were treated acutely with an endovascular stent graft (group 2). The resuscitation protocol and interval from onset of symptoms to treatment was comparable in both groups. Medical records were reviewed for prehospitalization and emergency department data, operative findings, and outcomes. RESULTS: There were 5 of 28 deaths (17.8%) in the surgical group and 1 of 26 deaths (3.8%) in the endovascular group. In the surgical group 1 of 28 patients (3.6%) exhibited paraplegia; there were no cases of paraplegia in the endovascular group. There were 4 of 28 cases (14.3%) of renal failure in group 1 and 1 of 26 (3.8%) in group 2. In group 1, 8 patients (28.6%) required mechanical ventilation for more than 48 hours; there were 2 of 26 patients (7.7%) in group 2 with this ventilatory requirement. Three patients required a repeat thoracotomy for hemorrhage in the surgical group. There were two access failures in the endovascular group. CONCLUSIONS: In the treatment of acute ruptures of the thoracic aorta, the immediate outcome of patients treated with endovascular stent grafts appears to be better than with management by conventional surgical repair.

Emergent endovascular stent grafting for perforated acute type B dissections and ruptured thoracic aortic aneurysms.

BACKGROUND: The purpose of our study was to demonstrate the effectiveness of endovascular stent grafts in the treatment of acutely ruptured thoracic aortic aneurysms and type B dissections as an alternative to the conventional surgical approach in an emergency setting. METHODS: From January 2001 to October 2001, we deployed 11 emergent endovascular stent grafts into the thoracic aorta. We treated seven ruptured aortic aneurysms and four acutely perforated type B dissections. Aortic rupture was confirmed preoperatively by spiral computed tomography. In all cases, hemothorax was present. The average interval from onset of symptoms to treatment was 28.5 hours. We used nine Talent and two Excluder stent grafts. RESULTS: Deployment of the stent grafts was successful in nine cases. There were two cases of access failure due to small caliber of iliac arteries, and 1 of these patients died shortly after the procedure was abandoned, At 12 months of follow-up, there were no cases of paraplegia, stent migration, or endoleaks. There was, however, one temporary renal failure, and 2 patients required mechanical ventilation for more than 48 hours. CONCLUSIONS: Our experiences with emergency endovascular stent grafting show that the procedure is technically feasible, with less morbidity and mortality than conventional open surgery, in high-risk patients.

Urgent thoracic aortal dissection and aneurysm: treatment with stent-graft implantation in an angiographic suite.

The aim of this study was to evaluate the feasibility of endoluminal stent-graft placement in an angiographic suite for the treatment of emergent type-B aortic dissections and ruptured thoracic aortal aneurysms. Twenty-six patients with either urgent type-B dissection (n=8) or aneurysms (n=18) of the descending thoracic aorta were chosen for stent-graft implantation. All patients received a multidetector-row CT angiography of the whole aorta and pelvic arteries prior to stent-graft implantation. All procedures were performed in a fully equipped digital subtraction angiography (DSA) suite under general anesthesia. In 20 patients Talent LPS tube grafts and in 4 patients an Excluder graft were used. Access was achieved via surgical cut-down in the left (n=7) or right (n=19) groin. Sealing was successful in 24 patients. The proximal covered portion of the stent graft was placed across the left subclavian artery in 2 patients. Procedural success was achieved in 23 of 24 patients. One patient required a second stent-graft placement before the aneurysm was sealed. One patient with an acute perforation of the descending aorta died due to cardiac failure prior to stent-graft implantation. In 1 patient stent-graft delivery failed due to severe calcification of both common iliac arteries. Endoluminal treatment of both urgent type-B aortic dissections and thoracic aortal aneurysms with stent graft is an attractive alternative treatment to surgical repair. The placement of stent grafts in an angiographic suite is a safe and feasible method with good clinical effectiveness and, so far, good clinical outcome.

Emergent endovascular interventions for contained rupture of thoracic aortic aneurysms.

BACKGROUND: The purpose of our study was to assess in an emergency setting the feasibility of endovascular stent graft treatment of contained ruptures of thoracic aortic aneurysms. METHODS: Seven patients with contained ruptures of thoracic aortic aneurysms from a series of 54 endovascular interventions were treated by the deployment of an aortic stent graft. In all cases, hemothorax was present. Acute deployment was performed with the patient under general anesthesia, and access was gained via the common femoral or iliac arteries. After a follow-up o f 12 months, the patients were evaluated by computed tomography. RESULTS: One patient died perioperatively. The procedure was technically successful in 5 patients. Perioperatively and at follow-up, there were no cases of paraplegia, stent graft migration, or endoleaks. There were, however, 2 cases of access failure, 1 case of temporary renal failure, and 2 patients who required prolonged mechanical ventilation. CONCLUSION: The acute treatment of contained ruptures of thoracic aortic aneurysms is feasible. This form of treatment seems to be a promising option in the treatment of these high-risk patients.

Surgical treatment of tracheomalacia by bronchoscopic monitored aortopexy in infants and children.

BACKGROUND: Aortopexy has become an established surgical procedure for the treatment of severe tracheomalacia (TM) in infancy. However, postoperative outcome may be improved by intraoperative bronchoscopic control of the aortopexy. METHODS: Between 1992 and 2000, 16 infants and children (2 female, 14 male) with TM were treated by pexis of the aorta via a right (15 patients) or left (1 patient) anterior thoracotomy. Patients age ranged from 4 to 122 months (mean, 26 mon). Three infants had previous surgery for esophagus atresia and tracheoesophageal fistula. Another four patients were operated for gastroesophageal reflux. In all cases, the aortopexy was monitored intraoperatively by bronchoscopy. Respiratory function was verified for each patient by comparing pre- and postoperative tidal expiratory flow values (TEF 25% in ml/sec). RESULTS: Mean follow-up was 36 months (range, 2 to 60 mo). There was no intraoperative or postoperative mortality. 13 patients showed permanent relief of symptoms. Postoperative median TEF 25% increased significantly compared with preoperative values (81 ml/sec vs. 56 ml/sec; p = 0.016). In one patient repeat aortopexy was necessary. CONCLUSIONS: Aortopexy through a right anterior thoracotomy is an efficient and feasible method in the surgical treatment of TM in infancy and, therefore, can improve postoperative respiratory function. Intraoperative bronchoscopy is advantageous.