The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study.

Introduction: Chronic rejection is a major complication post-transplantation. Within lung transplantation, chronic rejection was considered as airway centred. Chronic Lung Allograft Dysfunction (CLAD), defined to cover all late chronic complications, makes it more difficult to understand chronic rejection from an immunological perspective. This study investigated the true nature, timing and location of chronic rejection as a whole, within mouse lung transplantation. Methods: 40 mice underwent an orthotopic left lung transplantation, were sacrificed at day 70 and evaluated by histology and in vivo µCT. For timing and location of rejection, extra grafts were sacrificed at day 7, 35, 56 and investigated by ex vivo µCT or single cell RNA (scRNA) profiling. Results: Chronic rejection originated as innate inflammation around small arteries evolving toward adaptive organization with subsequent end-arterial fibrosis and obliterans. Subsequently, venous and pleural infiltration appeared, followed by airway related bronchiolar folding and rarely bronchiolitis obliterans was observed. Ex vivo µCT and scRNA profiling validated the time, location and sequence of events with endothelial destruction and activation as primary onset. Conclusion: Against the current belief, chronic rejection in lung transplantation may start as an arterial response, followed by responses in venules, pleura, and, only in the late stage, bronchioles, as may be seen in some but not all patients with CLAD.

Oxygenated machine perfusion at room temperature as an alternative for static cold storage in porcine donor hearts.

BACKGROUND: There is a continued interest in ex situ heart perfusion as an alternative strategy for donor heart preservation. We hypothesize that oxygenated machine perfusion of donor hearts at a temperature that avoids both normothermia and deep hypothermia offers adequate and safe preservation. METHODS: Cardioplegia-arrested porcine donor hearts were randomly assigned to six hours of preservation using cold storage (CS, n = 5) or machine perfusion using an oxygenated acellular perfusate at 21°C (MP, n = 5). Subsequently, all grafts were evaluated using the Langendorff method for 120 min. Metabolic parameters and histology were analyzed. Systolic function was assessed by contractility and elastance. Diastolic function was assessed by lusitropy and stiffness. RESULTS: For both groups, in vivo baseline and post-Langendorff biopsies were comparable, as were lactate difference and myocardial oxygen consumption. Injury markers gradually increased and were comparable. Significant weight gain was seen in MP (p = 0.008). Diastolic function was not impaired in MP, and lusitropy was superior from 30 min up to 90 min of reperfusion. Contractility was superior in MP during the first hour of evaluation. CONCLUSION: We conclude that the initial functional outcome of MP-preserved hearts was transiently superior compared to CS, with no histological injury post-Langendorff. Our machine perfusion strategy could offer feasible and safe storage of hearts prior to transplantation. Future studies are warranted for further optimization.

Defining and predicting progression in non-IPF interstitial lung disease.

Randomized placebo-controlled trials demonstrated the efficacy of antifibrotic treatment in non-IPF progressive fibrosing ILD (fILD). Currently, there is no consensus on how progression should be defined and clinical data of non-IPF fILD patients in a real-world setting are scarce. Non-IPF fILD patients presenting at the University Hospitals Leuven between 2012 and 2016 were included. Different definitions of progression according to the selection criteria of the INBUILD, RELIEF and the uILD study were retrospectively evaluated at every hospital visit. Univariate and multivariate analyses were performed to identify predictors of progression and mortality. The study cohort comprised 120 patients; 68.3%, 54.2% and 65.8% had progressive disease based on the INBUILD, RELIEF and uILD study, respectively. A large overlap of progressive fILD patients according to the different clinical trials was observed. Median transplant-free survival time of progressive fILD patients was 3.9, 3.9, 3.8 years and the median time-to-progression after diagnosis was 2.0, 3.1 and 2.3 years according to the INBUILD, RELIEF and uILD study, respectively. We identified several predictors of mortality, but only an underlying diagnosis of HP and uILD was independently associated with progression. Our data show a high prevalence of progressive fibrosis among non-IPF fILD patients and a discrepancy between predictors of mortality and progression. Mortality rate in fILD is high and the identification of progressive disease is only made late during the disease course. Moreover, future treatment decisions will be based upon disease behavior. Therefore, more predictors of progressive disease are needed to guide treatment decisions in the future.

Small airway loss in the physiologically ageing lung: a cross-sectional study in unused donor lungs.

BACKGROUND: Physiological lung ageing is associated with a gradual decline in dynamic lung volumes and a progressive increase in residual volume due to diminished elastic recoil of the lung, loss of alveolar tissue, and lower chest wall compliance. However, the effects of ageing on the small airways (ie, airways <2·0 mm in diameter) remain largely unknown. By using a combination of ex-vivo conventional CT (resolution 1 mm), whole lung micro-CT (resolution 150 µm), and micro-CT of extracted cores (resolution 10 µm), we aimed to provide a multiresolution assessment of the small airways in lung ageing in a large cohort of never smokers. METHODS: For this cross-sectional study, we included donor lungs collected from 32 deceased never-smoking donors (age range 16-83 years). Ex-vivo CT and whole lung high-resolution CT (micro-CT) were used to determine total airway numbers, stratified by airway diameter. Micro-CT was used to assess the number, length, and diameter of terminal bronchioles (ie, the last generation of conducting airways); mean linear intercept; and surface density in four lung tissue cores from each lung, extracted using a uniform sampling approach. Regression ß coefficients are calculated using linear regression and polynomial models. FINDINGS: Ex-vivo CT analysis showed an age-dependent decrease in the number of airways of diameter 2·0 mm to less than 2·5 mm (ß coefficient per decade -0·119, 95% CI -0·193 to -0·045; R2=0·29) and especially in airways smaller than 2·0 mm in diameter (-0·158, -0·233 to -0·084; R2=0·47), between 30 and 80 years of age, but not of the larger (≥2·5 mm) diameter airways (-0·00781, -0·04409 to 0·02848; R2=0·0007). In micro-CT analysis of small airways, the total number of terminal bronchioles per lung increased until the age of 30 years, after which an almost linear decline in the number of terminal bronchioles was observed (ß coefficient per decade -2035, 95% CI -2818 to -1252; R2=0·55), accompanied by a non-significant increase in alveolar airspace size (6·44, -0·57 to 13·45, R2=0·10). Moreover, this decrease in terminal bronchioles was associated with the age-related decline of pulmonary function predicted by healthy reference values. INTERPRETATION: Loss of terminal bronchioles is an important structural component of age-related decline in pulmonary function of healthy, non-smoking individuals. FUNDING: Research Foundation-Flanders, KU Leuven, Parker B Francis Foundation, UGent, Canadian Institutes for Health.

Small airways pathology in idiopathic pulmonary fibrosis: a retrospective cohort study.

BACKGROUND: The observation that patients with idiopathic pulmonary fibrosis (IPF) can have higher than normal expiratory flow rates at low lung volumes led to the conclusion that the airways are spared in IPF. This study aimed to re-examine the hypothesis that airways are spared in IPF using a multiresolution imaging protocol that combines multidetector CT (MDCT), with micro-CT and histology. METHODS: This was a retrospective cohort study comparing explanted lungs from patients with severe IPF treated by lung transplantation with a cohort of unused donor (control) lungs. The donor control lungs had no known lung disease, comorbidities, or structural lung injury, and were deemed appropriate for transplantation on review of the clinical files. The diagnosis of IPF in the lungs from patients was established by a multidisciplinary consensus committee according to existing guidelines, and was confirmed by video-assisted thoracic surgical biopsy or by pathological examination of the contralateral lung. The control and IPF groups were matched for age, sex, height, and bodyweight. Samples of lung tissue were compared using the multiresolution imaging approach: a cascade of clinical MDCT, micro-CT, and histological imaging. We did two experiments: in experiment 1, all the lungs were randomly sampled; in experiment 2, samples were selected from regions of minimal and established fibrosis. The patients and donors were recruited from the Katholieke Universiteit Leuven (Leuven, Belgium) and the University of Pennsylvania Hospital (Philadelphia, PA, USA). The study took place at the Katholieke Universiteit Leuven, and the University of British Columbia (Vancouver, BC, Canada). FINDINGS: Between Oct 5, 2009, and July 22, 2016, explanted lungs from patients with severe IPF (n=11), were compared with a cohort of unused donor (control) lungs (n=10), providing 240 samples of lung tissue for comparison using the multiresolution imaging approach. The MDCT specimen scans show that the number of visible airways located between the ninth generation (control 69 [SD 22] versus patients with IPF 105 [33], p=0·0023) and 14th generation (control 9 [6] versus patients with IPF 49 [28], p<0·0001) of airway branching are increased in patients with IPF, which we show by micro-CT is due to thickening of their walls and distortion of their lumens. The micro-CT analysis showed that compared with healthy (control) lung anatomy (mean 5·6 terminal bronchioles per mL [SD 1·6]), minimal fibrosis in IPF tissue was associated with a 57% loss of the terminal bronchioles (mean 2·4 terminal bronchioles per mL [SD 1·0]; p<0·0001), the appearance of fibroblastic foci, and infiltration of the tissue by inflammatory immune cells capable of forming lymphoid follicles. Established fibrosis in IPF tissue had a similar reduction (66%) in the number of terminal bronchioles (mean 1·9 terminal bronchioles per mL [SD 1·4]; p<0·0001) and was dominated by increased airspace size, Ashcroft fibrosis score, and volume fractions of tissue and collagen. INTERPRETATION: Small airways disease is a feature of IPF, with significant loss of terminal bronchioles occuring within regions of minimal fibrosis. On the basis of these findings, we postulate that the small airways could become a potential therapeutic target in IPF. FUNDING: Katholieke Universiteit Leuven, US National Institutes of Health, BC Lung Association, and Genentech.

The pleural mesothelium and transforming growth factor-ß1 pathways in restrictive allograft syndrome: A pre-clinical investigation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) hampers long-term survival after lung transplantation. Common fibrosis-related mechanisms in idiopathic pulmonary fibrosis and CLAD instigated the consideration of investigating the differential regulation of pleural mesothelium and transforming growth factor-ß1 (TGF-ß1) in restrictive allograft syndrome (RAS). METHODS: TGF-ß1 was assessed in bronchoalveolar lavage (BAL) fluid using enzyme-linked immunoassay and via immune staining of explant biopsies. To assess the role of the pleura, explanted bronchiolitis obliterans syndrome (BOS) and RAS lungs were compared using computed tomography scans, calretinin stainings, Western blot, and quantititative real-time PCR. Last, a pleural mesothelial cell line was used to assess mesothelial-to-mesenchymal transition and its inhibition. RESULTS: TGF-ß1 was increased in BAL of RAS patients (p = 0.035), and was present in the (sub)pleural area of biopsies. Explanted RAS lungs demonstrated an increased volume fraction of pleura (p = 0.0004), a higher proportion of calretinin-positive stainings (p = 0.0032), and decreased E-cadherin (p = 0.019) and increased α-smooth muscle actin (p = 0.0089) mRNA expression and protein levels in isolated pleural tissue. Moreover, TGF-ß1 stimulation of pleural mesothelial cells led to a phenotypical switch to mesenchymal cells, accompanied with an increased migratory capacity. Interleukin-1α was able to accentuate TGF-ß1‒induced mesothelial-to-mesenchymal transition. None of the tested drugs could inhibit mesothelial-to-mesenchymal transition at the used concentrations. CONCLUSIONS: Our results support an interplay between TGF-ß1 and the pleural mesothelium in the pathophysiology of RAS.

Role of 18F-FDG PET/CT in Restrictive Allograft Syndrome After Lung Transplantation.

BACKGROUND: Differential diagnosis of phenotypes of chronic lung allograft dysfunction (CLAD) remains troublesome. We hypothesized that F-fluorodeoxyglucose positron emission tomography with computed tomography (F-FDG PET/CT) may help in differential diagnosis of CLAD phenotypes, as it showed promising results regarding diagnosis and prognosis in interstitial lung diseases. METHODS: A monocentric, retrospective study was performed including all lung transplant recipients suffering from bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) who underwent F-FDG PET/CT scan, in comparison with stable lung transplant recipients. Maximum standardized uptake value (SUVmax) was associated with pulmonary function and survival. Proof-of-concept microCT and glucose transporter-1 staining served as morphologic validation for regions with different SUVmax. RESULTS: Maximum standardized uptake value was higher in RAS (median, 2.6; n = 29) compared with BOS (median, 1.0; n = 15) and stable patients (median, 0.59; n = 8) (P < 0.0001). In RAS, high SUVmax was associated with worse survival after F-FDG PET/CT (P = 0.0004; hazard ratio, 1.82). Forced vital capacity at F-FDG PET/CT inversely correlated with SUVmax (R = -0.40, P = 0.03). MicroCT analysis revealed extensive fibrosis in regions of high SUVmax, with an increased number of glucose transporter-1-positive cells. CONCLUSIONS: F-fluorodeoxyglucose positron emission tomography with CT may noninvasively differentiate RAS from BOS. RAS patients with areas of increased lung metabolism have worse outcome, demonstrating the potential use of F-FDG PET/CT during follow-up after lung transplantation.

The histomorphological spectrum of restrictive chronic lung allograft dysfunction and implications for prognosis.

Chronic lung allograft dysfunction continues to be the main contributor to poor long-term allograft survival after lung transplantation. The restrictive phenotype of chronic lung allograft dysfunction carries a particularly poor prognosis. Little is known about the pathogenetic mechanisms involved in restrictive chronic lung allograft dysfunction. In this study, we performed histomorphological and immunohistochemical analysis of restrictive chronic lung allograft dysfunction lungs. Explant lung tissue from 21 restrictive chronic lung allograft dysfunction patients was collected and histopathologic patterns of rejection, fibrosis and vascular changes were scored after routine histochemical stains and additional immunohistochemistry for endothelial markers and C4d. In all, 75% of cases showed evidence of acute cellular rejection; lymphocytic bronchiolitis was absent in most lungs, whereas in 55% there was obliterative bronchiolitis. Almost half of the cases showed a pattern consistent with pleuroparenchymal fibro-elastosis (n=10), and a subset showed nonspecific interstitial pneumonia (n=5) or irregular emphysema (n=5). Fibrinous alveolar exudates were frequently seen in association with fibrosis (n=6), but no diffuse alveolar damage was found. Evidence of microvascular damage was present in most cases. An emphysematous pattern of fibrosis was associated with a better survival (P=0.0030), whereas fibrinous exudates were associated with a worse survival (P=0.0007). In addition to the previously described nonspecific interstitial pneumonia and pleuroparenchymal fibro-elastosis patterns in restrictive chronic lung allograft dysfunction, we are the first to describe a pattern of fibrosis-induced subpleural/paraseptal emphysema. This pattern confers a better survival, whereas fibrinous exudates are associated with a worse survival. We believe that our findings offer a pathogenetic theory for pleuroparenchymal fibro-elastosis in restrictive chronic lung allograft dysfunction, and show that restrictive chronic lung allograft dysfunction is an increasingly heterogeneous disease with presumably different mechanisms of subpattern formation.

End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterised by vigorous airway inflammation eventually resulting in severe lung damage. This study aimed to describe the diversity of the inflammatory pattern in end-stage CF lungs by evaluating and quantifying which components of the innate and adaptive immunity are involved, and by assessing whether this is gender-specific. METHODS: CF explant lung tissue (n = 20) collected at time of transplantation and control tissue (n = 22) was sectioned (9 µm) and stained for neutrophils, eosinophils, mast cells, dendritic cells, macrophages, CD4 T cells, cytotoxic T cells and B cells. Quantification with special attention for immune cell location was performed. RESULTS: Neutrophils, mast cells, dendritic cells, macrophages, CD4 T and cytotoxic T cells were significantly increased in CF compared to controls and there was a disproportionate increase of neutrophils around the airways in CF. Large amounts of lymphoid follicles were found in the CF lung and they had a skewed B cell/T cell composition. Upon subdividing the CF patients into a male and female population, eosinophils, mast cells and CD4 T cells were increased specifically in CF females. In this subpopulation, lymphoid follicles had less B cells and more CD8 T cells. CONCLUSION: These data demonstrate a diverse inflammatory response in the CF lung, reflected by an increase of both myeloid and lymphoid immune cells. Inflammation in the CF lung appeared to be gender-specific in our population, as the significant increase of eosinophils, mast cells and CD4 T cells was especially notable in the female subpopulation.

Immunological diversity in phenotypes of chronic lung allograft dysfunction: a comprehensive immunohistochemical analysis.

Chronic rejection after organ transplantation is defined as a humoral- and cell-mediated immune response directed against the allograft. In lung transplantation, chronic rejection is nowadays clinically defined as a cause of chronic lung allograft dysfunction (CLAD), consisting of different clinical phenotypes including restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome (BOS). However, the differential role of humoral and cellular immunity is not investigated up to now. Explant lungs of patients with end-stage BOS (n = 19) and RAS (n = 18) were assessed for the presence of lymphoid (B and T cells) and myeloid cells (dendritic cells, eosinophils, mast cells, neutrophils, and macrophages) and compared to nontransplant control lung biopsies (n = 21). All myeloid cells, with exception of dendritic cells, were increased in RAS versus control (neutrophils, eosinophils, and mast cells: all P < 0.05, macrophages: P < 0.001). Regarding lymphoid cells, B cells and cytotoxic T cells were increased remarkably in RAS versus control (P < 0.001) and in BOS versus control (P < 0.01). Interestingly, lymphoid follicles were restricted to RAS (P < 0.001 versus control and P < 0.05 versus BOS). Our data suggest an immunological diversity between BOS and RAS, with a more pronounced involvement of the B-cell response in RAS characterized by a structural organization of lymphoid follicles. This may impact future therapeutic approaches.

Tracheal tissue-engineering: in-vivo biocompatibility of mechanically-stripped allogenic rabbit trachea with autologous epithelial covering.

BACKGROUND: Successful trachea transplantation comprises the use of biocompatible constructs with little immune-reactivity, submucosal revascularization and creation of an epithelial covering. Allogenic chondrocytes might be protected from an overt immune-response due to physical isolation. Our aim was to evaluate in-vivo biocompatibility of allotracheae, stripped of their highly-immunogenic inner lining. Secondly, we established whether these constructs might serve as suitable scaffolds for autologous epithelial grafting. METHODS: Mucosa and submucosa of 12 rabbit donor tracheae were mechanically peeled off. Cartilage was covered with Integra™ regeneration-template. Constructs were implanted within the recipient's lateral thoracic artery flap. Integra of 6 revascularized allotracheae was grafted with autologous buccal mucosa. Macroscopical, histological analysis and immunohistochemistry were performed. RESULTS: Revascularization and buccal grafting was incomplete in the first 2 circular constructs. To enhance blood-vessel outgrowth, the following 10 transplants were opened longitudinally before implantation. Integra revascularized well. Grafted tracheae showed satisfactory mucosa-adherence, albeit with invasion of migrating epithelium within the Integra-scaffold. CONCLUSIONS: Mechanically-stripped allotracheae exhibited beneficial biocompatibility up to two months. This approach might open doors in the treatment of long-segment tracheal pathologies of which immunosuppression is contra-indicated. Thickness of this layered construct limited practical feasibility of orthotopic transfer, though with further refinements, a clinically-useful transplant could be created.

Morphometric Analysis of Explant Lungs in Cystic Fibrosis.

RATIONALE: After repeated cycles of lung infection and inflammation, patients with cystic fibrosis (CF) evolve to respiratory insufficiency. Although histology and imaging have provided descriptive information, a thorough morphometric analysis of end-stage CF lung disease is lacking. OBJECTIVES: To quantify the involvement of small and large airways in end-stage CF. METHODS: Multidetector computed tomography (MDCT) and micro-CT were applied to 11 air-inflated CF explanted lungs and 7 control lungs to measure, count, and describe the airway and parenchymal abnormalities in end-stage CF lungs. Selected abnormalities were further investigated with thin section histology. MEASUREMENTS AND MAIN RESULTS: On MDCT, CF explanted lungs showed an increased median (interquartile range) number (631 [511-710] vs. 344 [277-349]; P = 0.003) and size of visible airways (cumulative airway diameter 217 cm [209-250] vs. 91 cm [80-105]; P < 0.001) compared with controls. Airway obstruction was seen, starting from generation 6 and increasing to 40 to 50% of airways from generation 9 onward. Micro-CT showed that the total number of terminal bronchioles was decreased (2.9/ml [2.6-4.4] vs. 5.3/ml [4.8-5.7]; P < 0.001); 49% were obstructed, and the cross-sectional area of the open terminal bronchioles was reduced (0.093 mm(2) [0.084-0.123] vs. 0.179 mm(2) [0.140-0.196]; P < 0.001). On micro-CT, 41% of the obstructed airways reopened more distally. This remodeling was confirmed on histological analysis. Parenchymal changes were also seen, mostly in a patchy and peribronchiolar distribution. CONCLUSIONS: Extensive changes of dilatation and obstruction in nearly all airway generations were observed in end-stage CF lung disease.

Linking clinical phenotypes of chronic lung allograft dysfunction to changes in lung structure.

Chronic lung allograft dysfunction (CLAD) remains the major barrier to long-term success after lung transplantation. This report compares gross and microscopic features of lungs removed from patients receiving a redo-transplant as treatment for CLAD. Lungs donated by patients with either the bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) phenotype of CLAD and appropriate control lungs (eight per group) were air-inflated, frozen solid and kept frozen while a multi-detector computed tomography (MDCT) was obtained. The lung was then cut into 2-cm thick transverse slices and sampled for micro-CT and histopathology. The MDCT showed reduced lung volume with increased lung weight and density in RAS versus BOS and control (p<0.05). Although pre-terminal bronchioles were obstructed in both phenotypes, RAS lungs showed a reduction of pre-terminal bronchioles (p<0.01). Micro-CT and matched histopathology showed that RAS was associated with reduced numbers of terminal bronchioles/lung compared to BOS and controls (p<0.01), with expansion of the interstitial compartment and obliteration of the alveolar airspaces by fibrous connective tissue. RAS is associated with greater destruction of both pre-terminal and terminal bronchioles. Additionally, the interstitial compartments are expanded and alveolar airspaces are obliterated by accumulation of fibrous connective tissue.

The site and nature of airway obstruction after lung transplantation.

RATIONALE: The chronic rejection of lung allografts is attributable to progressive small airway obstruction. OBJECTIVES: To determine precisely the site and nature of this type of airway obstruction. METHODS: Lungs from patients with rejected lung allografts treated by a second transplant (n = 7) were compared with unused donor (control) lungs (n = 7) using multidetector computed tomography (MDCT) to determine the percentage of visible airways obstructed in each airway generation, micro-computed tomography (microCT) to visualize the site of obstruction, and histology to determine the nature of this obstruction. MEASUREMENTS AND MAIN RESULTS: The number of airways visible with MDCT was not different between rejected and control lungs. However, 10 ± 7% of observed airways greater than 2 mm in diameter, 50 ± 22% of airways between 1 and 2 mm in diameter, and 73 ± 10% of airways less than 1 mm in diameter were obstructed in the rejected lungs. MicroCT confirmed that the mean lumen diameter of obstructed airways was 647 ± 317 µm but showed no difference in either total number and cross-sectional area of the terminal bronchioles or in alveolar dimensions (mean linear intercept) between groups (P > 0.05). In addition, microCT demonstrated that only segments of the airways are obstructed. Histology confirmed a constrictive form of bronchiolitis caused by expansion of microvascular-rich granulation tissue in some locations and collagen-rich scar tissue in others. CONCLUSIONS: Chronic lung allograft rejection is associated with a progressive form of constrictive bronchiolitis that targets conducting airways while sparing larger airways as well as terminal bronchioles and the alveolar surface.

Partial mechanical circulatory support in an ovine model of post-infarction remodeling.

BACKGROUND: Full unloading of the left ventricle (LV) in chronic heart failure (CHF) induces reversal of LV dilation and geometric distortion. In this study we describe the partial unloading effects in ischemic CHF. METHODS: Six weeks after myocardial infarction, sheep were randomized to partial support ("pump," n = 5), as provided by the CircuLite Synergy micro-pump, or to no therapy ("sham," n = 6) for an additional 6 weeks. At baseline, and at 6 and 12 weeks after infarction, pressure-volume (PV) recordings were made. Systolic and diastolic functions were characterized by the end-systolic volume (ESV) where LV end-systolic pressure reached 90 mm Hg (V90), and the end-diastolic volume (EDV) where LV end-diastolic pressure reached 15 mm Hg (V15), respectively. Magnetic resonance imaging (MRI) was performed 6 and 12 weeks after infarction. During autopsy at 12 weeks, isolated LVs were weighed. Histologically, the degree of fibrosis in the non-infarcted area was assessed using systematic randomized sampling, and myocyte hypertrophy was measured by the mean linear intercept method. RESULTS: At 6 weeks, PV measurements showed a V90 and V15 increase (p = NS between groups). Six weeks later, V90 and V15 increased in the sham group. In the pump group, V90 decreased but V15 did not change significantly. At 6 weeks, MRI indicated no significant difference between groups. Six weeks later, in the sham group, EDV and ESV increased significantly. In the pump group, EDV decreased significantly and ESV trended to decrease. Sphericity index increased in the sham group and decreased in the pump group, although not significantly. Explanted LV masses were significantly higher in the sham group than in the pump group. The pump group had a decrease in fibrosis and less myocyte hypertrophy. CONCLUSION: Partial support 6 weeks after major myocardial infarction halts and reverses ventricular dilation and hypertrophy.

Involvement of interleukin-17 during lymphocytic bronchiolitis in lung transplant patients.

BACKGROUND: Interleukin-17 (IL-17) is involved in autoimmune and chronic pulmonary diseases and linked with neutrophilic inflammation. Azithromycin reduces and prevents broncholaveolar lavage (BAL) neutrophilia after lung transplantation (LTx). In this investigation we assessed the involvement of IL-17 in different post-transplant complications in human LTx biopsies. METHODS: Immunohistochemical staining against IL-17A was performed on biopsies of LTx patients with either chronic rejection, acute A-grade rejection (A > 2B0), lymphocytic bronchiolitis (LB), infection, and stable patients. Biopsies of 7 patients with LB were stained before and after azithromycin treatment. IL-17+ cells were quantified as number per square millimeter of lamina propria. Double staining with CD4/CD8 was performed to determine the origin of IL-17. RESULTS: In the LB group, biopsies showed a significant presence of IL-17+ cells/mm2 of lamina propria compared with the stable, acute A-grade/chronic rejection and infection groups (p < 0.0001). The number of IL-17+ cells on biopsy correlated with percent BAL (%BAL) neutrophilia (r = 0.34, p = 0.0056). Azithromycin reduced both %BAL neutrophilia and IL-17+ cells (both p = 0.016) in the LB group. CD8+ cells were the major source of IL-17. CONCLUSIONS: IL-17+ / CD8+ cells are present in LB after LTx but not in acute A-grade/chronic rejection nor during infection. Moreover, azithromycin significantly decreased the number of IL-17+ cells in the airway wall, which may further explain its effect on BAL neutrophilia.

Long-term nose-only cigarette smoke exposure induces emphysema and mild skeletal muscle dysfunction in mice.

Mouse models of chronic obstructive pulmonary disease (COPD) focus on airway inflammation and lung histology, but their use has been hampered by the lack of pulmonary function data in their assessment. Systemic effects such as muscle dysfunction are also poorly modeled in emphysematous mice. We aimed to develop a cigarette-smoke-induced emphysema mouse model in which serial lung function and muscular dysfunction could be assessed, allowing the disease to be monitored more appropriately. C57Bl6 mice were nose-only exposed to cigarette smoke or filtered air for 3-6 months. Lung function tests were repeated in the same mice after 3 and 6 months of cigarette smoke or air exposure and compared with lung histological changes. Contractile properties of skeletal muscles and muscle histology were also determined at similar time points in separate groups of mice. Serial lung function measurements documented hyperinflation after 3 and 6 months of cigarette smoke exposure, with a significant 31-37% increase in total lung capacity (TLC) and a significant 26-35% increase in compliance (Cchord) when compared with animals exposed to filtered air only (P<0.001 after 3 and after 6 months). These functional changes preceded the changes in mean linear intercept, which became only significant after 6 months of cigarette smoke exposure and which correlated very well with TLC (r=0.74, P=0.004) and Cchord (r=0.79, P=0.001). After 6 months of cigarette smoke exposure, a significant fiber-type shift from IIa to IIx/b was also observed in the soleus muscle (P<0.05), whereas a 20% reduction of force was present at high stimulation frequencies (80 Hz; P=0.09). The extensor digitorum longus (EDL) muscle was not affected by cigarette smoke exposure. These serial pulmonary function variables are sensitive outcomes to detect emphysema progression in a nose-only cigarette-smoke-exposed animal model of COPD. In this model, muscular changes became apparent only after 6 months, particularly in muscles with a mixed fiber-type composition.

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research.

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(©) - FlexiVent or Buxco(©) - Forced Pulmonary Maneuvers(®) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.

Innate and adaptive interleukin-17-producing lymphocytes in chronic inflammatory lung disorders.

During T-cell receptor activation in a particular cytokine environment, naive CD4+ T cells may differentiate into lineages defined by their pattern of cytokine production and transcription factors: T helper type 1 (Th1), Th2, Th17, and Th22 cells; follicular helper T cells; and inducible regulatory T cells. Th17 cells have been recognized as a distinct lineage of Th cells, and associations between IL-17 and human disease have been known somewhat longer. It would be an oversimplification to restrict IL-17 to Th17 cells. Indeed, IL-17 is also expressed by other cells including IL-17-producing γδ T (γδ T-17) cells, natural killer T-17 cells, and IL-17-producing lymphoid tissue-induced cells. IL-17 was cloned in 1995 as a cytokine expressed by T cells, exerting inflammatory effects on epithelial, endothelial, and fibroblast cells. IL-17 is a solid link between innate and adaptive immunity and can exert both beneficial and deleterious effects. The discovery of IL-17 T cells has provided exciting new insights into host defense, immunoregulation, and autoimmunity. Unquestionably, data from mouse models have contributed enormously to our insight into immunological mechanisms. However, because of numerous differences between murine and human immunology, data obtained in mice are not simply interchangeable. We review IL-17 T cells exclusively in the human situation and more specifically their potential role in respiratory diseases. The advances in our understanding of IL-17 regulation offer opportunities to dissect the human IL-17 system and to reflect on the clinical presentation of lung diseases. More importantly, the IL-17 system allows us to speculate on new therapeutic opportunities. Some results have been previously reported in an abstract.

Bronchoalveolar lavage neutrophilia in acute lung allograft rejection and lymphocytic bronchiolitis.

BACKGROUND: Acute cellular rejection and lymphocytic bronchiolitis can impair allograft function after lung transplant (LTx). Both may be refractory to corticosteroid treatment. We hypothesized that bronchoalveolar lavage (BAL) neutrophilia may be increased in either acute rejection or lymphocytic bronchiolitis or may increase with increasing histologic severity. METHODS: All consecutive BAL with subsequent transbronchial biopsy (TBB) specimens, performed in 339 LTx recipients from 2001 to 2008, were retrospectively analyzed. TBB specimens were classified according to histologic grade with analysis of BAL total cell count and cell differentials. RESULTS: The analysis included 768 TBB specimens. After adjustment for possible confounders, BAL total cell count significantly increased both with grade A or B severity (p < 0.0001). A higher A grade was characterized by a significant increase in BAL lymphocytosis and neutrophilia (p < 0.0001), whereas for higher B grades, only a more prominent BAL neutrophilia was seen (p < 0.0001). CONCLUSIONS: Higher grade A, but, particularly, higher grade B severity scores are characterized by increased BAL neutrophilia.