Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

BACKGROUND: Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. METHODS: We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. RESULTS: Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. CONCLUSIONS: The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

Derivation of induced pluripotent stem cells from ferret somatic cells.

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Depletion of Airway Submucosal Glands and TP63+KRT5+ Basal Cells in Obliterative Bronchiolitis.

RATIONALE: Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB. OBJECTIVES: Two major SC compartments in airways are submucosal glands (SMGs) and surface airway p63 (also known as TP63 [tumor protein 63])-positive/K5 (also known as KRT5 [keratin 5])-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB. METHODS: Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay. MEASUREMENTS AND MAIN RESULTS: Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63+/K5+ and increase in K5+/K14+ and K14+ BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model. CONCLUSIONS: SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.

A brain leptin-renin angiotensin system interaction in the regulation of sympathetic nerve activity.

The sympathetic nervous system, leptin, and renin-angiotensin system (RAS) have been implicated in obesity-associated hypertension. There is increasing evidence for the presence of both leptin and angiotensin II receptors in several key brain cardiovascular and metabolic control regions. We tested the hypothesis that the brain RAS plays a facilitatory role in the sympathetic nerve responses to leptin. In rats, intracerebroventricular (ICV) administration of losartan (5 µg) selectively inhibited increases in renal and brown adipose tissue (BAT) sympathetic nerve activity (SNA) produced by leptin (10 µg ICV) but did not reduce the SNA responses to corticotrophin-releasing factor (CRF) or the melanocortin receptor agonist MTII. In mice with deletion of angiotensin II type-1a receptors (AT(1a)R(-/-)), increases in renal and BAT SNA induced by leptin (2 µg ICV) were impaired whereas SNA responses to MTII were preserved. Decreases in food intake and body weight with ICV leptin did not differ in AT(1a)R(-/-) vs. AT(1a)R(+/+) mice. ICV leptin in rats increased AT(1a)R and angiotensin-converting enzyme (ACE) mRNA in the subfornical organ and AT(1a)R mRNA in the arcuate nucleus, suggesting leptin-induced upregulation of the brain RAS in specific brain regions. To evaluate the role of de novo production of brain angiotensin II in SNA responses to leptin, we treated rats with captopril (12.5 µg ICV). Captopril attenuated leptin effects on renal and BAT SNA. In conclusion, these studies provide evidence that the brain RAS selectively facilitates renal and BAT sympathetic nerve responses to leptin while sparing effects on food intake.

Perfluorocarbon induced intra-arrest hypothermia does not improve survival in a swine model of asphyxial cardiac arrest.

BACKGROUND: Pulseless electrical activity is an important cause of cardiac arrest. Our purpose was to determine if induction of hypothermia with a cold perfluorocarbon-based total liquid ventilation (TLV) system would improve resuscitation success in a swine model of asphyxial cardiac arrest/PEA. METHODS: Twenty swine were randomly assigned to control (C, no ventilation, n=11) or TLV with pre-cooled PFC (n=9) groups. Asphyxia was induced by insertion of a stopper into the endotracheal tube, and continued in both groups until loss of aortic pulsations (LOAP) was reached, defined as a pulse pressure less than 2mmHg. The TLV animals underwent asphyxial arrest for an additional 2min after LOAP, followed by 3min of hypothermia, prior to starting CPR. The C animals underwent 5min of asphyxia beyond LOAP. Both groups then underwent CPR for at least 10min. The endpoint was the resumption of spontaneous circulation maintained for 10min. RESULTS: Seven of 9 animals achieved resumption of spontaneous circulation (ROSC) in the TLV group vs. 5 of 11 in the C group (p=0.2). The mean pulmonary arterial temperature was lower in total liquid ventilation animals starting 4min after induction of hypothermia (TLV 36.3+/-0.2 degrees C vs. C 38.1+/-0.2 degrees C, p<0.0001). Arterial P(O)(2) was higher in total liquid ventilation animals at 2.5min of CPR (TLV 76+/-12mmHg vs. C 44+/-2mmHg; p=0.03). CONCLUSION: Induction of moderate hypothermia using perfluorocarbon-based total liquid ventilation did not improve ROSC success in this model of asphyxial cardiac arrest.

Selecting the transthoracic defibrillation shock directional vector based on VF amplitude improves shock success.

INTRODUCTION: Termination of ventricular fibrillation (VF) by a defibrillating shock is more likely to occur when the VF amplitude is larger. We hypothesized that a defibrillation shock would achieve higher success if the shock vector was oriented along the largest of the VF amplitudes measured simultaneously in 3 orthogonal ECG leads, and that this axis could be determined near-instantaneously in real time. METHODS AND RESULTS: In 9 closed-chest anesthetized swine, a new directional defibrillation (DD) device was used to simultaneously measure the VF peak amplitudes displayed by 3 orthogonal pairs of defibrillation electrodes: anterior-posterior, lateral-lateral, and superior-inferior. Four shocks at each of 3 energy levels (30 Joules [J], 50 J, and 100 J) were delivered through the electrode pair measuring the largest (LA) and smallest (SA) VF peak amplitude at the time of the shock. The odds of shock success (VF termination followed by a perfusing rhythm) were 5 times more likely when shocks were delivered from the LA electrodes than the SA electrodes (odds ratio 5.10, 95% CI: 1.39, 18.79). At the intermediate energy level of 50 J, shocks delivered through the LA electrode pairs had an almost 9 times higher odds of shock success than 50 J shocks delivered through the SA electrode pairs (68.3% vs 18.9%, P = 0.002) (odds ratio 8.94, 95% CI: 2.59, 30.82). Transthoracic impedance and current did not differ for shocks delivered in the LA versus SA groups. CONCLUSION: Choosing the defibrillation directional vector based on the largest VF amplitude improved shock success.

Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation.

BACKGROUND: Rapid intra-arrest induction of hypothermia using total liquid ventilation (TLV) with cold perfluorocarbons improves resuscitation outcome from ventricular fibrillation (VF). Cold saline intravenous infusion during cardiopulmonary resuscitation (CPR) is a simpler method of inducing hypothermia. We compared these 2 methods of rapid hypothermia induction for cardiac resuscitation. METHODS: Three groups of swine were studied: cold preoxygenated TLV (TLV, n=8), cold intravenous saline infusion (S, n=8), and control (C, n=8). VF was electrically induced. Beginning at 8 min of VF, TLV and S animals received 3 min of cold TLV or rapid cold saline infusion. After 11 min of VF, all groups received standard air ventilation and closed chest massage. Defibrillation was attempted after 3 min of CPR (14 min of VF). The end point was resumption of spontaneous circulation (ROSC). RESULTS: Pulmonary arterial (PA) temperature decreased after 1 min of CPR from 37.2 degrees C to 32.2 degrees C in S and from 37.1 degrees C to 34.8 degrees C in TLV (S or TLV vs. C p<0.0001). Coronary perfusion pressure (CPP) was higher in TLV than S animals during the initial 3 min of CPR. Arterial pO(2) was higher in the preoxygenated TLV animals. ROSC was achieved in 7 of 8 TLV, 2 of 8 S, and 1 of 8C (TLV vs. C, p=0.03). CONCLUSIONS: Moderate hypothermia was achieved rapidly during VF and CPR using both cold saline infusion and cold TLV, but ROSC was higher than control only in cold TLV animals, probably due to better CPP and pO(2). The method by which hypothermia is achieved influences ROSC.

Liquid ventilation with perfluorocarbons facilitates resumption of spontaneous circulation in a swine cardiac arrest model.

BACKGROUND: Induced external hypothermia during ventricular fibrillation (VF) improves resuscitation outcomes. Our objectives were twofold (1) to determine if very rapid hypothermia could be achieved by intrapulmonary administration of cold perfluorocarbons (PFC), thereby using the lungs as a vehicle for targeted cardiopulmonary hypothermia, and (2) to determine if this improved resuscitation success. METHODS: Part 1: Nine female swine underwent static intrapulmonary instillation of cold perfluorocarbons (PFC) during electrically induced VF. Part 2: Thirty-three female swine in VF were immediately ventilated via total liquid ventilation (TLV) with pre-oxygenated cold PFC (-15 degrees C) or warm PFC (33 degrees C), while control swine received no ventilation during VF. All swine in both Parts 1 and 2 underwent VF arrest for 11 min, then defibrillation, ventilation and closed chest massage until resumption of spontaneous circulation (ROSC). The endpoint was continued spontaneous circulation for 1h without pharmacologic support. RESULTS: Static intrapulmonary instillation of cold PFC achieved rapid cardiopulmonary hypothermia; pulmonary artery (PA) temperature of 33.5+/-0.2 degrees C was achieved by 10 min. Nine of 9 achieved ROSC. Hypothermia was achieved faster using TLV: at 6 min VF, cold TLV temperature was 32.9+/-0.4 degrees C vs. cold static instillation temperature 34.3+/-0.2 degrees C. Nine of 11 cold TLV swine achieved ROSC for 1h vs. 3 of 11 control swine (p=0.03). Warm PFC also appeared to be beneficial, with a trend toward greater achievement of ROSC than control (ROSC; warm PFC 8 of 11 vs. control 3 of 11, p=0.09). CONCLUSION: Targeted cardiopulmonary intra-arrest moderate hypothermia was achieved rapidly by static intrapulmonary administration of cold PFC and more rapidly by total liquid ventilation with cold PFC; resumption of spontaneous circulation was facilitated. Warm PFC showed a trend toward facilitating ROSC.

Serological methods and selective agars to enumerate Campylobacter from broiler carcasses: data from inter- and intralaboratory analyses.

Routine analytical means to estimate Campylobacter numbers per milliliter of carcass rinses are needed in high-sample-throughput poultry laboratories. We compared three serological confirmatory tests that were amenable to such a setting when used in conjunction with Campy-Line and Campy-Cefex Campylobacter selective agars. Pre- and post-chlorinated chiller carcass rinse samples were obtained and held on ice, then analyzed 24 h later in two separate laboratories. Presumptive counts on both pre- and postchiller samples from between laboratories on individual agars and between both agars were highly correlated. Agreement among the three serological tests was nearly complete. The use of a premeasured and dried latex anti-Campylobacter antibody agglutination test format was superior to that of either a liquid latex agglutination format or a direct phosphate-buffer microscopic technique in terms of practicality as was the inclusion of an unarmed latex control to detect auto agglutination. A routine procedure for Campylobacter level estimation was suggested. This procedure, when used in conjunction with a serological confirmatory step, should provide processors with a means to assess reductions in numbers per milliliter of carcass rinses versus strictly presence-absence testing.