Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder.

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.

A Novel Technique of Long-Segment Tracheal Repair With Extended Bronchial Flap of Right Upper and Main Bronchus Plus Tracheoplasty.

Long-segment tracheal resection and repair pose a great challenge. We present a successful case of long-segment tracheal defect repair with extended bronchial flap of the right upper and main bronchus, together with a tracheoplastic method. This is a novel technique for repairing a large tracheal defect with an extended pedicled bronchial flap in specific situations.

Bronchoplastic closure as an alternative approach for tracheal reconstruction following resection of a massive tracheal tumour.

A 47-year old woman presented with large cell carcinoma with extensive lengthwise and circumferential invasion of the lower trachea. End-to-end anastomosis by suture lines alone may be impossible and even harmful, following tumour resection with such extensive tracheal involvement. Thus, we performed a successful tracheal reconstruction with bronchoplastic closure without complications or recurrence at 12-month follow-up. This case highlights the use of this technique for the closure of massive airway defects.

Allogeneic CD4+CD25high T cells regulate obliterative bronchiolitis of heterotopic bronchus allografts in both porcinized and humanized mouse models.

BACKGROUND: Bronchiolitis obliterans syndrome is caused by a fibroproliferative process in lung allografts resulting in irreversible damage. In this study, we induced obliterative bronchiolitis and studied the contribution of regulatory T cells to its development in immune-deficient mice receiving heterotopic porcine bronchus transplants, and major histocompatibility complex-mismatched porcine peripheral blood mononuclear cell. Furthermore, we aimed to corroborate our findings in a humanized mouse model. METHODS: Heterotopic bronchus transplantation was performed in 33 NOD.rag(−/−)γc(−/−) mice, using miniature pigs as tissue donors.The recipient mice then either received saline (negative control), unsorted MHC-mismatched PBMC (positive control), PBMC enriched with CD4(+)CD25(high) cells or PBMC depleted of CD4(+)CD25(high) cells for reconstitution. The results were validated in 28 NOD.rag(−/−)γc(−/−) mice undergoing heterotopic human bronchus transplantation and reconstitution with allogeneic human PBMC. RESULTS: Histological lesions similar to those typical for obliterative bronchiolitis developed in vivo after reconstitution with allogeneic PBMC and were more severe in animals engrafted with PBMC depleted of CD4(+)CD25(high) cells. In contrast, the group reconstituted with PBMC enriched with CD4(+)CD25(high) cells showed well-preserved histology. The results of the humanized model confirmed those obtained in the porcinized model. CONCLUSIONS: In conclusion, both porcinized and humanized mouse models of heterotopic subcutaneous bronchus transplantation imitate the in vivo development of bronchiolitis obliterans syndrome-like lesions and reveal its sensitivity to T-cell regulation.

Salvaging the dehisced lung transplant bronchial anastomosis with homograft aorta.

This is a case of 50-year-old male who underwent left single lung transplantation for pulmonary fibrosis. He sustained a bronchial dehiscence with a pulmonary artery-bronchial fistula which was primarily repaired. One week later, there was complete bronchial dehiscence followed by a massive hemoptysis. At operation, following resection of necrotic donor bronchus there was a sizeable gap between donor and recipient bronchus, which was bridged with a cryopreserved aortic homograft. The homograft patch provided a satisfactory repair without malacia. The patient required retransplantation six months later for reasons unassociated with the repair. Homograft aorta proved useful material for salvaging the dehisced lung transplant bronchial anastomosis.

Ingraft chimerism in lung transplantation--a study in a porcine model of obliterative bronchiolitis.

BACKGROUND: Bronchial epithelium is a target of the alloimmune response in lung transplantation, and intact epithelium may protect allografts from rejection and obliterative bronchiolitis (OB). Herein we study the influence of chimerism on bronchial epithelium and OB development in pigs. METHODS: A total of 54 immunosuppressed and unimmunosuppressed bronchial allografts were serially obtained 2-90 days after transplantation. Histology (H&E) was assessed and the fluorescence in situ hybridization (FISH) method for Y chromosomes using pig-specific DNA-label was used to detect recipient derived cells in graft epithelium and bronchial wall, and donor cell migration to recipient organs. Ingraft chimerism was studied by using male recipients with female donors, whereas donor cell migration to recipient organs was studied using female recipients with male donors. RESULTS: Early appearance of recipient-derived cells in the airway epithelium appeared predictive of epithelial destruction (R=0.610-0.671 and p<0.05) and of obliteration of the bronchial lumen (R=0.698 and p<0.01). All allografts with preserved epithelium showed epithelial chimerism throughout the follow-up. Antirejection medication did not prevent, but delayed the appearance of Y chromosome positive cells in the epithelium (p<0.05), or bronchial wall (p<0.05). CONCLUSIONS: In this study we demonstrate that early appearance of Y chromosomes in the airway epithelium predicts features characteristic of OB. Chimerism occurred in all allografts, including those without features of OB. Therefore we suggest that ingraft chimerism may be a mechanism involved in the repair of alloimmune-mediated tissue injury after transplantation.

Kergin pneumonectomy: a rare procedure.

We have evaluated our cases of "Kergin" pneumonectomy in which a bronchial flap of the medial part of the right main stem bronchus was created during right pneumonectomy and was turned upward to cover the lateral airway defect at the level of the carina. Five male patients with a mean age of 53.6 underwent "Kergin" pneumonectomy due to nonsmall cell carcinoma arising from right upper lobe entrance, which does not allow a classical pneumonectomy. Postoperative pathology stagings were stage IIB in 1 patient and stage IIIA in 4 patients. Any operative mortality or short-term complication was not observed. Two of the patients died in the second year of follow-up. "Kergin" pneumonectomy is a rarely performed procedure with acceptable morbidity and mortality and good lung cancer resection. Actually, our current report of five cases will be one of the largest series of "Kergin" pneumonectomy.

Bronchial flap closure of the lower membranous trachea.

A novel method for closure of the lower membranous trachea after right pneumonectomy using a flap derived from the cartilaginous portion of the right main bronchus is described in this study. This technique was used successfully in patients with tracheal stenosis due to a giant posterior mediastinal tumor known as schwannoma. Because of the severe tracheobronchial stenosis and destroyed right lung, tumor resection combined with resection of the lower membranous trachea and right pneumonectomy was carried out. We closed the defect in the membranous lower trachea with the flap derived from the right main bronchus. The clinical course was uneventful.

Reconstruction of the lower trachea using a pedicled autologous bronchial flap.

We describe a technique used in a patient for resection of adenoid cystic carcinoma arising from the left main bronchus and extending along the lateral wall of the lower trachea without carinal invasion. A flap was mobilized from the noninvolved lateral wall of the left main bronchus, which was left attached to the carina. This was used to close the defect in the lower trachea with the implantation of the left main bronchus, avoiding a tracheal sleeve pneumonectomy. Clinical course of this case was good. The pedicled autologous bronchial flap provides reliable material to repair and reconstruct a massive central airway defect.

Local C-reactive protein expression in obliterative lesions and the bronchial wall in posttransplant obliterative bronchiolitis.

The local immunoreactivity of C-reactive protein (CRP) was studied in a heterotopic porcine model of posttranplant obliterative bronchiolitis (OB). Bronchial allografts and control autografts were examined serially 2-28 days after subcutaneous transplantation. The autografts stayed patent. In the allografts, proliferation of inflammatory cells (P < .0001) and fibroblasts (P = .02) resulted in occlusion of the bronchial lumens (P < .01). Influx of CD4+ (P < .001) and CD8+ (P < .0001) cells demonstrated allograft immune response. CRP positivity simultaneously increased in the bronchial walls (P < .01), in macrophages, myofibroblasts, and endothelial cells. Local CRP was predictive of features characteristic of OB (R = 0.456-0.879, P < .05-P < .0001). Early obliterative lesions also showed CRP positivity, but not mature, collagen-rich obliterative plugs (P < .05). During OB development, CRP is localized in inflammatory cells, myofibroblasts and endothelial cells probably as a part of the local inflammatory response.

Epithelial tenascin predicts obliterative airway disease.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis and airway obliteration result in remodeling of terminal bronchi in post-transplant obliterative bronchiolitis. Tenascin as an extracellular matrix glycoprotein is expressed in several remodeling processes. METHODS: Heterotopic bronchial allografts of pigs were studied to assess tenascin expression during development of post-transplant obliterative bronchiolitis. A total of 157 allografts or autograft controls were serially obtained 2 to 28 days after transplantation and processed for histology and immunocytochemistry for tenascin, CD4, CD8 and macrophages. Epithelial tenascin index was calculated by multiplying the percentage of positive cells by the grade of tenascin intensity (1 to 3). RESULTS: Epithelial tenascin expression occurred during the initial ischemic damage to the respiratory epithelium. After partial recovery and before total epithelial loss and subsequent airway obliteration, tenascin expression peaked in allografts (p < 0.001). Epithelial tenascin index on Day 7 was predictive of subsequent epithelial damage, bronchial wall inflammation and the number of (CD4(+) and CD8(+)) cells, fibroproliferation, and obliteration of the bronchial lumen (R > or = 0.47, p < or = 0.01). Tenascin expression in the bronchial wall was more intense in allografts (p < 0.001), paralleling proliferation of fibroblasts and influx of inflammatory cells, and was predictive of inflammatory alterations also in the early obliterative lesions (R > or = 0.45, p < 0.05). Expression decreased during maturation of fibrosis (p < 0.05). CONCLUSIONS: Epithelial tenascin was predictive of features observed in post-transplant obliterative bronchiolitis, demonstrating a role for tenascin in the development of obliterative bronchiolitis. Tenascin may have relevant properties in serving as a clinical marker for early obliterative bronchiolitis.

Thoracoscopic right middle lobar bronchus reimplantation for iatrogenic injury in infant: is it a good option?

We report the first case in the literature of thoracoscopic bronchial reimplantation in 5 month-old boy. The child was born with congenital cystic adenomatoid malformation of the right lower lobe associated with lower intralobar pulmonary sequestration diagnosed prenatally. An iatrogenic middle lobar bronchus injury was detected per-operatively during thoracoscopic lobectomy. Management and follow-up were exposed.

Platelet-derived growth factor, transforming growth factor-beta, and connective tissue growth factor in a porcine bronchial model of obliterative bronchiolitis.

The expression of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, and connective tissue growth factor (CTGF) and the effect of imatinib, an agent inhibiting PDGF receptors, were assessed in a porcine bronchial transplantation model of obliterative bronchiolitis (OB). Up-regulation of PDGF-A, PDGF receptors alpha and beta, and TGF-beta expression occurred in allografts, whereas PDGF-B and CTGF expression was similar in allo- and autografts. Imatinib modified the inflammatory responses and expression patterns of PDGF-A and PDGF receptors. This study further confirms PDGF and TGF-beta as mediators of OB and supports the concept of the importance of the pathways signaled through PDGF receptors in post-transplant OB.

Tenascin is expressed in post-transplant obliterative bronchiolitis.

Tenascin is expressed in inflammatory and fibroproliferative processes, both contributing to posttransplant obliterative bronchiolitis (OB) in association with epithelial cell injury and airway obliteration. We studied bronchial allografts to elucidate the role of tenascin during this alloimmune response. Bronchial segments were subcutaneously implanted into eight pigs. Allografts and autograft controls were serially obtained until total obliteration in allografts and processed for histology and immunocytochemistry for CD4, CD8, and tenascin. Findings were graded on a scale from 0 to 3. In autografts the operative epithelial damage recovered and bronchi stayed patent with mild-graded fibrosis and inflammation. Partial recovery was observed in allografts on day 4, thereafter the epithelial loss gradually increased. Total recovery was achieved on day 11 (P < .001). Fibroblast proliferation resulted in total luminal obliteration on day 21 (P < .001). The number of inflammatory cells increased rapidly (P < .05) with numerous CD4+ and CD8+ cells on day 14 (P < .0001). Prior to total epithelial loss in allografts, tenascin expression was observed on day 7 in 69% of epithelial cells, whereas in only 5% of epithelial cells in recovered autografts. Paralleling the most intense fibroproliferation, tenascin-positive cells were observed in the bronchial wall on day 7 and day 11 (P < .001). Tenascin expression was demonstrated during the inflammatory response and fibroblast proliferation during the early stage of obliterative bronchiolitis showing that tenascin contributes to posttransplant obliterative bronchiolitis development.

Immune cells in a heterotopic lamb-to-pig bronchial xenograft model.

We developed our porcine model to elucidate the cellular rejection mechanisms of xenografts. Bronchial segments from a donor lamb were implanted into domestic pigs. The immunosuppressive regimens consisted of no immusuppression, or of daily oral cyclosporine A (CsA) 15 mg/kg, or of everolimus, 1.5 mg/kg, or of both. Implants were serially harvested during 17 days. Epithelial damage and obliteration were graded histologically, followed by a count of CD4+, CD8+, MHC class II-expressing cells, and macrophages. Furthermore, we studied the pharmocokinetics of everolismus. Epithelial damage preceded luminal obliteration, which was eventually total, except when both drugs had been given. In xenografts, an influx of cells with CD8+ cells dominating peaked on day 9, thereafter declining, except in the combination drug group. There, the immunological reaction was delayed and blunted, with CD4+ cells dominating. More macrophages appeared in xenografts than in allografts except with the combination CsA and everolimus. A dose of 1.5 mg/kg everolimus yields adequate blood concentrations for porcine studies. In this xenograft model, chronic rejection appears to be caused by an immune response to the graft, but it is more short-lived than the response in allografts. The combination of CsA and everolimus was able to blunt the response and delay the subsequent obliteration.

Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces.

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus ("normal" model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated ("thick") mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. METHODS: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies. RESULTS: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. CONCLUSIONS: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Cyclooxygenase-2 expression in experimental post-transplant obliterative bronchiolitis.

Epithelial cell injury, inflammation, progressive fibrosis, and airway obliteration are histological features of post-transplant obliterative bronchiolitis (OB). Cyclooxygenase (COX)-2 is expressed in acute and chronic inflammatory responses. Our aim was to elucidate the possible role of COX-2 in post-transplant OB by using a heterotopic bronchial porcine model. Bronchial allografts from non-related donors were transplanted subcutaneously into 24 random-bred domestic pigs, each weighing about 20 kg. Groups studied had grafts, non-treated allografts, allografts given cyclosporine A (CsA), methylprednisolone (MP), and azathioprine (Aza), and allografts given CsA, MP, and everolimus. Grafts were serially harvested during a follow-up period of 21 days for histology (H&E) and immunohistochemistry. Immunostaining was performed with monoclonal IgG against human COX-2 peptide, and histological alterations and immunohistochemical positivity were graded on a scale from 0 to 5. Epithelial COX-2 index was calculated by multiplying the percentage of positive cells by grade of epithelial COX-2 intensity. Ischaemic epithelial loss, evident in all implants, recovered rapidly in autografts, and bronchi remained patent. Epithelial loss in non-treated allografts preceded fibroblast proliferation, resulting in total luminal obliteration. In CsA-, MP-, and Aza-treated allografts epithelial destruction and luminal obliteration were delayed, and these were prevented in CsA-, MP-, and everolimus-treated allografts. COX-2 expression due to operative ischaemia was evident in all implants on day 2. Thereafter, the epithelial COX-2 index preceded epithelial injury and obliteration. During the inflammatory response and fibroblast proliferation, COX-2 expression occurred in macrophages and fibroblasts. In conclusion, in the early stage of OB development, COX-2 induction occurred in airway epithelial cells prior to luminal obliteration. In addition, the observation that COX-2 expression in macrophages and fibroblasts paralleled the onset of inflammation and fibroblast proliferation indicates a role in OB development, but the causal relationships need further study.

Heterotopic en bloc tracheobronchial transplantation with direct revascularization in pigs.

OBJECTIVE: This article describes the application of a novel aortic tube technique for directly revascularized tracheobronchial transplantation with dual blood supply in pigs. METHODS: Eleven adult Large White pigs underwent heterotopic tracheal transplantation with a dual revascularization technique (inferior thyroid artery and bronchial artery). Seven tracheobronchial grafts were perfused ex vivo, and hemodynamic data were collected. RESULTS: At the last evaluation, 6 pigs had normally epithelialized mucus-producing allografts with correct morphologic conformation and cartilage viability. The histopathologic examination revealed homogeneous tissue regardless of biopsy site (trachea, carina, or bronchi), demonstrating the efficacy of the revascularization procedure. Four animals had early ischemic necrosis develop, 2 from acute rejection and 2 from technical mishap. One additional pig had acute rejection starting on the 14th postoperative day. The CD4(+)/CD8(+) ratio was maintained close to or above 0.8 in the subgroup with rejection and below 0.6 in the animals that were correctly immunosuppressed. Pressure-flow curves in 7 ex vivo tracheobronchial grafts showed a nonsignificant difference (P <.12) in vascular resistance between the bronchial artery territory (lower resistance) and the inferior thyroid artery territory. CONCLUSIONS: For the first time, a transplantation technique encompassing the entire trachea, carina, and stem bronchi has been made possible. By means of the dual inferior thyroid and bronchial artery axis, we were able to obtain a structurally healthy and functional graft to replace the main airway.