Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

Lung Xenotransplantation.

Although efforts have been made to expand the pool of donor lung allografts for human lung transplantation, a shortage remains. Lung xenotransplantation has been proposed as an alternative approach, but lung xenotransplantation in humans has not yet been reported. In addition, significant biological and ethical barriers will have to be addressed before clinical trials can be undertaken. However, significant progress has been made toward addressing biological incompatibilities that present a barrier, and recent advances in genetic engineering tools promise to accelerate further progress.

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.

Timing of Esophagectomy after Neoadjuvant Chemoradiation Therapy Affects the Incidence of Anastomotic Leaks.

BACKGROUND: Neoadjuvant chemoradiation therapy (nCRT) has become the standard of care for esophageal cancer patients prior to esophagectomy. However, the optimal timing for surgery after completion of nCRT remains unclear. METHODS: A retrospective review was performed of patients who underwent esophagectomy with cervical anastomosis for esophageal cancer at a single institution between January 2000 and June 2015. Patients were categorized into 3 cohorts: those who did not receive nCRT prior to esophagectomy (no nCRT), those who underwent esophagectomy within 35 days after nCRT (≤35d), and those who underwent esophagectomy more than 35 days after nCRT (>35d). RESULTS: A total of 366 esophagectomies were performed during the study period, and 348 patients met the inclusion criteria. Anastomotic leaks occurred in 11.8% of all patients included in the study (41 of 348). Within each cohort, anastomotic leaks were detected in 14.7% of patients (17 of 116) in the no nCRT cohort, 7.3% (13 of 177) in the ≤35d cohort, and 20.0% (11 of 55) in the >35d cohort (p=0.020). Significant differences in the occurrence of anastomotic leaks were observed between the no nCRT and ≤35d cohorts (p=0.044), and between the ≤35d and >35d cohorts (p=0.007). CONCLUSION: Esophagectomy with cervical anastomosis within 35 days of nCRT resulted in a lower percentage of anastomotic leaks.

Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus.

The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 "safe harbor" locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG-LoxPtdTomatoStopLoxPEGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.

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.

Sporadic hybrid oncocytic/chromophobe tumor of the kidney: a clinicopathologic, histomorphologic, immunohistochemical, ultrastructural, and molecular cytogenetic study of 14 cases.

Hybrid oncocytic/chromophobe tumors (HOCT) of the kidney have been described in patients with Birt-Hogg-Dubé syndrome (BHD) and in association with renal oncocytosis without BHD. HOCT in patients without evidence of BHD or renal oncocytosis is exceedingly rare, and these cases have been poorly characterized. We have identified and studied 14 cases of HOCT from previously diagnosed renal oncocytomas (398 cases) and chromophobe renal cell carcinomas (351 cases) without evidence of BHD or renal oncocytosis. Immunohistochemical, ultrastructural, and molecular genetic studies analyzing numerical chromosomal changes, loss of heterozygosity for chromosome 3p, and mutation status of VHL, c-kit, PDGFR, and folliculin (FLCN) genes were performed. HOCTs were identified in nine men and five women (age range 40-79 years). The size of tumors ranged from 2 to 11 cm. All tumors displayed a solid alveolar architecture and were composed of cells with abundant granular eosinophilic oncocytic cytoplasm with perinuclear halos. Occasional binucleated neoplastic cells were present, but irregular, hyperchromatic, wrinkled (raisinoid) nuclei were absent. The cytoplasm contained numerous mitochondria of varying sizes, but only sparse microvesicles with amorphic lamellar content were found. Tumors were positive for CK7 (12/14), AE1-AE3 (14/14), anti-mitochondrial antigen (14/14), E-cadherin (11/13), parvalbumin (12/14), and epithelial membrane antigen (14/14). Tumors were generally negative for racemase, CK20, CD10, and carboanhydrase IX. Interphase fluorescence in situ hybridization revealed multiple chromosomal losses and gains with a median of four (range 1-9) chromosomal aberrations per case. Monosomy of chromosome 20 was common and found in 7 of 14 cases. Monosomy of chromosomes 6 and 9 was present in 4 of 14 cases each, of which two cases displayed monosomy for both chromosomes 6 and 9. Polysomy of chromosomes 10, 21, and 22 was found in 4/14 cases each, of which one case displayed polysomy for all these three chromosomes. No pathogenic mutations were found in the VHL, c-kit, PDGFR, and folliculin (FLCN) genes. (1) We have shown that hybrid oncocytic/chromophobe tumors of the kidney do occur, albeit rarely, outside the Birt-Hogg-Dubé syndrome and without associated renal oncocytosis. (2) These tumors constitute a relatively homogenous group with histomorphologic features of both chromophobe renal cell carcinoma and renal oncocytoma. (3) Sporadic hybrid oncocytic/chromophobe renal tumors are characterized by multiple numerical aberrations (both mono- and polysomies) of chromosomes 1, 2, 6, 9, 10, 13, 17, 21, and 22 and lack of mutations in the VHL, c-kit, PDGFRA, and FLCN genes. (4) The tumors seem to behave indolently as no evidence of malignant behavior was documented in our series, although admittedly, the follow-up was too short to fully elucidate the biological nature of this rare neoplasm. At worst, these tumors could have a low malignant potential, which only can be found out with longer follow-up.