Microscopic Small Airway Abnormalities Identified in Early Idiopathic Pulmonary Fibrosis In Vivo Using Endobronchial Optical Coherence Tomography.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) affects subpleural lung, but is considered to spare small airways. Micro-CT studies demonstrated small airway reduction in end-stage IPF explanted lungs, raising questions about small airway involvement in early-stage disease. Endobronchial optical coherence tomography (EB-OCT) is a volumetric imaging modality that detects microscopic features from subpleural to proximal airways. We use EB-OCT to evaluate small airways in early IPF and control subjects in vivo. METHODS: EB-OCT was performed in 12 IPF and 5 control subjects (matched by age, sex, smoking-history, height, BMI). IPF subjects had early disease with mild restriction (FVC: 83.5% predicted), diagnosed per current guidelines and confirmed by surgical biopsy. EB-OCT volumetric imaging was acquired bronchoscopically in multiple, distinct, bilateral lung locations (total: 97 sites). IPF imaging sites were classified by severity into affected (all criteria for UIP present) and less affected (some but not all criteria for UIP present) sites. Bronchiole count and small airway stereology metrics were measured for each EB-OCT imaging site. RESULTS: Compared to control subjects (mean: 11.2 bronchioles/cm3; SD: 6.2), there was significant bronchiole reduction in IPF subjects (42% loss; mean: 6.5/cm3; SD: 3.4; p=0.0039), including in IPF affected (48% loss; mean: 5.8/cm3; SD: 2.8; p<0.00001) and IPF less affected (33% loss; mean: 7.5/cm3; SD: 4.1; p=0.024) sites. Stereology metrics showed IPF affected small airways were significantly larger and more distorted/irregular than in IPF less affected sites and control subjects. IPF less affected and control airways were statistically indistinguishable for all stereology parameters (p=0.36-1.0). CONCLUSION: EB-OCT demonstrated marked bronchiolar loss in early IPF (between 30 and 50%), even in areas minimally affected by disease, compared to matched controls. These findings support small airway disease as a feature of early IPF, providing novel insight into pathogenesis and potential therapeutic targets.

Diagnostic Accuracy of Endobronchial Optical Coherence Tomography for the Microscopic Diagnosis of Usual Interstitial Pneumonia.

Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and DlCO were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8-100.0%) and 100% (79.6-100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72-1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.

Human-scale lung regeneration based on decellularized matrix scaffolds as a biologic platform.

Lung transplantation is currently the only curative treatment for patients with end-stage lung disease; however, donor organ shortage and the need for intense immunosuppression limit its broad clinical application. Bioartificial lungs created by combining native matrix scaffolds with patient-derived cells might overcome these problems. Decellularization involves stripping away cells while leaving behind the extracellular matrix scaffold. Cadaveric lungs are decellularized by detergent perfusion, and histologic examination confirms the absence of cellular components but the preservation of matrix proteins. The resulting lung scaffolds are recellularized in a bioreactor that provides biomimetic conditions, including vascular perfusion and liquid ventilation. Cell seeding, engraftment, and tissue maturation are achieved in whole-organ culture. Bioartificial lungs are transplantable, similarly to donor lungs, because the scaffolds preserve the vascular and airway architecture. In rat and porcine transplantation models, successful anastomoses of the vasculature and the airway were achieved, and gas exchange was evident after reperfusion. However, long-term function has not been achieved because of the immaturity of the vascular bed and distal lung epithelia. The goal of this strategy is to create patient-specific transplantable lungs using induced pluripotent stem cell (iPSC)-derived cells. The repopulation of decellularized scaffolds to create transplantable organs is one of possible future clinical applications of iPSCs.

Image-guided Preoperative Localization of Pulmonary Nodules for Video-assisted and Robotically Assisted Surgery.

Video-assisted thoracic surgery (VATS) and robotically assisted surgery are used increasingly for minimally invasive diagnostic and therapeutic resection of pulmonary nodules. Unsuccessful localization of small, impalpable, or deep pulmonary nodules can necessitate conversion from VATS to open thoracotomy. Preoperative localization techniques performed by radiologists have improved the success rates of VATS resection for small and subsolid nodules. Any center at which VATS diagnostic resection of indeterminate pulmonary nodules is performed should be supported by radiologists who offer preoperative nodule localization. Many techniques have been described, including image-guided injection of radioisotopes and radiopaque liquids and placement of metallic wires, coils, and fiducial markers. These markers enable the surgeon to visualize the position of an impalpable nodule intraoperatively. This article provides details on how to perform each percutaneous localization technique, and a group of national experts with established nodule localization programs describe their preferred approaches. Special reference is made to equipment required, optimization of marker placement, prevention of technique-specific complications, and postprocedural treatment. This comprehensive unbiased review provides valuable information for those who are considering implementation or optimization of a nodule localization program according to workflow patterns, surgeon preference, and institutional resources in a particular center. ©RSNA, 2019.

Bioengineering Human Lung Grafts on Porcine Matrix.

OBJECTIVE: Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix. SUMMARY BACKGROUND DATA: Implantable bioartificial organ grafts could revolutionize transplant surgery. To date, several milestones toward that goal have been achieved in rodent models. To make bioengineered organ grafts clinically relevant, scaling to human cells and graft size are the next steps. METHODS: We seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from rejected donor lungs, and banked human umbilical vein endothelial cells. We subsequently enabled tissue formation in whole organ culture. The resulting grafts were then either analyzed in vitro (n = 15) or transplanted into porcine recipients in vivo (n = 3). RESULTS: By repopulating porcine extracellular matrix scaffolds with human endothelial cells, we generated pulmonary vasculature with mature endothelial lining and sufficient anti-thrombotic function to enable blood perfusion. By repopulating the epithelial surface with human epithelial progenitor cells, we created a living, functioning gas exchange graft. After surgical implantation, the bioengineered lung grafts were able to withstand physiological blood flow from the recipient's pulmonary circulation, and exchanged gases upon ventilation during the 1-hour observation. CONCLUSIONS: Engineering and transplantation of viable lung grafts based on decellularized porcine lung scaffolds and human endothelial and epithelial cells is technically feasible. Further graft maturation will be necessary to enable higher-level functions such as mucociliary clearance, and ventilation-perfusion matching.

Can We Re-Engineer the Endocrine Pancreas?

PURPOSE OF REVIEW: Engineering endocrine pancreatic tissue is an emerging topic in type 1 diabetes with the intent to overcome the current limitation of ß cell transplantation. During islet isolation, the vascularized structure and surrounding extracellular matrix (ECM) are completely disrupted. Once implanted, islets slowly engraft and mostly are lost for the initial avascular phase. This review discusses the main building blocks required to engineer the endocrine pancreas: (i) islet niche ECM, (ii) islet niche vascular network, and (iii) new available sources of endocrine cells. RECENT FINDINGS: Current approaches include the following: tissue engineering of endocrine grafts by seeding of native or synthetic ECM scaffolds with human islets, vascularization of native or synthetic ECM prior to implantation, vascular functionalization of ECM structures to enhance angiogenesis after implantation, generation of engineered animals as human organ donors, and embryonic and pluripotent stem cell-derived endocrine cells that may be encapsulated or genetically engineered to be immunotolerated. Substantial technological improvements have been made to regenerate or engineer endocrine pancreatic tissue; however, significant hurdles remain, and more research is needed to develop a technology to integrate all components of viable endocrine tissue for clinical application.

Bioengineering Human Myocardium on Native Extracellular Matrix.

RATIONALE: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. OBJECTIVE: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes. METHODS AND RESULTS: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. CONCLUSIONS: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.

Development of a large-volume human-derived adipose acellular allogenic flap by perfusion decellularization.

In reconstructive surgery, transfer of patients' tissue (autologous flaps) is routinely used to repair large soft tissue defects caused by surgery, trauma, chronic diseases, or malformations; unfortunately, this strategy is not always possible and often creates a secondary defect in the donor site of the tissue. Tissue-engineered synthetic flaps are currently unable to repair clinically-relevant, large-volume defects; allogenic flaps from cadaveric donors could provide a ready-to-use biological alternative if treated with methods to avoid the immune-rejection of the donor's cells. Here, we describe the successful decellularization of a large (> 800 cc) human-derived adipose flap through a perfusion apparatus; we demonstrate the complete removal of the immunogenic cellular components of the flap with the retention of its structural components and vascular network. Our aim is to obtain a universally compatible, off-the-shelf acellular allogenic flap that could be recellularized with cells from recipient patients to provide a tissue-engineered allogenic/autologous alternative for reconstruction of large-volume soft-tissue defects.

Cell therapy for lung diseases. Report from an NIH-NHLBI workshop, November 13-14, 2012.

The National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health convened the Cell Therapy for Lung Disease Working Group on November 13-14, 2012, to review and formulate recommendations for future research directions. The workshop brought together investigators studying basic mechanisms and the roles of cell therapy in preclinical models of lung injury and pulmonary vascular disease, with clinical trial experts in cell therapy for cardiovascular diseases and experts from the NHLBI Production Assistance for Cell Therapy program. The purpose of the workshop was to discuss the current status of basic investigations in lung cell therapy, to identify some of the scientific gaps in current knowledge regarding the potential roles and mechanisms of cell therapy in the treatment of lung diseases, and to develop recommendations to the NHLBI and the research community on scientific priorities and practical steps that would lead to first-in-human trials of lung cell therapy.

Recellularization of organs: what is the future for solid organ transplantation?

PURPOSE OF REVIEW: Allogeneic organ transplantation is burdened by donor shortage, graft rejection and adverse effects of lifelong immune suppression. Engineering bioartificial organs from acellular organ scaffolds and patient-derived cells are a new approach to potentially overcome these limitations. RECENT FINDINGS: Decellularized organs yield a scaffold of extracellular matrix on which cells can adhere, integrate and ultimately form functional tissue. Various cell sources are currently used to repopulate acellular scaffolds, however, all have limitations. Patient-derived pluripotent stem cells hold great promise for tissue and organ engineering, when robust and mature cells can be directed in a reliable and safe manner. Finally, to produce mature organotypic tissue from a nonfunctional seeded scaffold, cellular scaffolds are cultured under biomimetic conditions in vitro. Alternatively, organs may be implanted at an immature stage to harness the recipient's body's regenerative capacity. In proof of principle experiments to date, bioengineered small animal organs have shown rudimentary function and maintained patency for limited time when transplanted in vivo. SUMMARY: Recent advances in bioengineering organs raise the hope that we can overcome organ donor shortage and eliminate the need for livelong immunosuppression. However, significant challenges remain in generating mature large-scale donor-like bioartificial organs.

Decellularized scaffolds as a platform for bioengineered organs.

PURPOSE OF REVIEW: Patients suffering from end-stage organ failure requiring organ transplantation face donor organ shortage and adverse effect of chronic immunosuppression. Recent progress in the field of organ bioengineering based on decellularized organ scaffolds and patient-derived cells holds great promise to address these issues. RECENT FINDINGS: Perfusion-decellularization is the most consistent method to obtain decellularized whole-organ scaffolds to serve as a platform for organ bioengineering. Important advances have occurred in organ bioengineering using decellularized scaffolds in small animal models. However, the function exhibited by bioengineered organs has been rudimentary. Pluripotent stem cells seem to hold promise as the ideal regenerative cells to be used with this approach but the techniques to effectively and reliably manipulate their fate are still to be discovered. Finally, this technology needs to be scaled up to human size to be of clinical relevance. SUMMARY: The search for alternatives to allogeneic organ transplantation continues. Important milestones have been achieved in organ bioengineering with the use of decellularized scaffolds. However, many challenges remain on the way to producing an autologous, fully functional organ that can be transplanted similar to a donor organ.

Intratracheally injected human-induced pluripotent stem cell-derived pneumocytes and endothelial cells engraft in the distal lung and ameliorate emphysema in a rat model.

OBJECTIVES: Pulmonary emphysema is characterized by the destruction of alveolar units and reduced gas exchange capacity. In the present study, we aimed to deliver induced pluripotent stem cell-derived endothelial cells and pneumocytes to repair and regenerate distal lung tissue in an elastase-induced emphysema model. METHODS: We induced emphysema in athymic rats via intratracheal injection of elastase as previously reported. At 21 and 35 days after elastase treatment, we suspended 80 million induced pluripotent stem cell-derived endothelial cells and 20 million induced pluripotent stem cell-derived pneumocytes in hydrogel and injected the mixture intratracheally. On day 49 after elastase treatment, we performed imaging, functional analysis, and collected lungs for histology. RESULTS: Using immunofluorescence detection of human-specific human leukocyte antigen 1, human-specific CD31, and anti--green fluorescent protein for the reporter labeled pneumocytes, we found that transplanted cells engrafted in 14.69% ± 0.95% of the host alveoli and fully integrated to form vascularized alveoli together with host cells. Transmission electron microscopy confirmed the incorporation of the transplanted human cells and the formation of a blood-air barrier. Human endothelial cells formed perfused vasculature. Computed tomography scans revealed improved vascular density and decelerated emphysema progression in cell-treated lungs. Proliferation of both human and rat cell was higher in cell-treated versus nontreated controls. Cell treatment reduced alveolar enlargement, improved dynamic compliance and residual volume, and improved diffusion capacity. CONCLUSIONS: Our findings suggest that human induced pluripotent stem cell-derived distal lung cells can engraft in emphysematous lungs and participate in the formation of functional distal lung units to ameliorate the progression of emphysema.

B cell-dependent subtypes and treatment-based immune correlates to survival in stage 3 and 4 lung adenocarcinomas.

Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA-Seq datasets. As previously described by others, pre-treatment expression of intratumoral 12-chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre-treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non-ICI treatments.

A quality improvement study on avoidable stressors and countermeasures affecting surgical motor performance and learning.

OBJECTIVE: To explore how the 2 most important components of surgical performance--speed and accuracy-are influenced by different forms of stress and what the impact of music is on these factors. BACKGROUND: On the basis of a recently published pilot study on surgical experts, we designed an experiment examining the effects of auditory stress, mental stress, and music on surgical performance and learning and then correlated the data psychometric measures to the role of music in a novice surgeon's life. METHODS: Thirty-one surgeons were recruited for a crossover study. Surgeons were randomized to 4 simple standardized tasks to be performed on the SurgicalSIM VR laparoscopic simulator (Medical Education Technologies, Inc, Sarasota, FL), allowing exact tracking of speed and accuracy. Tasks were performed under a variety of conditions, including silence, dichotic music (auditory stress), defined classical music (auditory relaxation), and mental loading (mental arithmetic tasks). Tasks were performed twice to test for memory consolidation and to accommodate for baseline variability. Performance was correlated to the brief Musical Experience Questionnaire (MEQ). RESULTS: Mental loading influences performance with respect to accuracy, speed, and recall more negatively than does auditory stress. Defined classical music might lead to minimally worse performance initially but leads to significantly improved memory consolidation. Furthermore, psychologic testing of the volunteers suggests that surgeons with greater musical commitment, measured by the MEQ, perform worse under the mental loading condition. CONCLUSIONS: Mental distraction and auditory stress negatively affect specific components of surgical learning and performance. If used appropriately, classical music may positively affect surgical memory consolidation. It also may be possible to predict surgeons' performance and learning under stress through psychological tests on the role of music in a surgeon's life. Further investigation is necessary to determine the cognitive processes behind these correlations.

Liquid Ventilation Reconditions Isolated Rat Lungs Following Ischemia-Reperfusion Injury.

Lung transplantation remains the only curative treatment for end-stage pulmonary disease. Lung ischemia-reperfusion injury (IRI) is a major contributor to primary allograft dysfunction and donor organ nonutilization. The alveolar macrophage is a key inflammatory mediator in IRI. Ex vivo lung perfusion (EVLP) has been investigated to rehabilitate lungs before transplant but has failed to provide significant improvements after IRI. We hypothesized that liquid ventilation (LV) could be utilized for ex vivo lung reconditioning in a rat IRI model. We compared EVLP with LV in an isolated ex vivo rat lung with an aqueous ventilant using quantitative physiological and immunological parameters. We observed improved physiological parameters and mechanical clearance of alveolar macrophages and cytokines halting the propagation of the inflammatory response in IRI. While the wide applicability to large animal or human transplantation have yet to be explored, these findings represent a method for lung reconditioning in the setting of significant IRI that could widen the lung organ donation pool and limit morbidity and mortality associated with ischemia-induced primary graft dysfunction.

Protease inhibitor Camostat Mesyalte blocks wild type SARS-CoV-2 and D614G viral entry in human engineered miniature lungs.

The catastrophic global effects of the SARS-CoV-2 pandemic highlight the need to develop novel therapeutics strategies to prevent and treat viral infections of the respiratory tract. To enable this work, we need scalable, affordable, and physiologically relevant models of the human lung, the primary organ involved in the pathogenesis of COVID-19. To date, most COVID-19 in vitro models rely on platforms such as cell lines and organoids. While 2D and 3D models have provided important insights, human distal lung models that can model epithelial viral uptake have yet to be established. We hypothesized that by leveraging techniques of whole organ engineering and directed differentiation of induced pluripotent stem cells (iPSC) we could model human distal lung epithelium, examine viral infection at the tissue level in real time, and establish a platform for COVID-19 related research ex vivo. In the present study, we used type 2 alveolar epithelial cells (AT2) derived from human iPSCs to repopulate whole rat lung acellular scaffolds and maintained them in extended biomimetic organ culture for 30 days to induce the maturation of distal lung epithelium. We observed emergence of a mixed type 1 and type 2 alveolar epithelial phenotype during tissue formation. When exposing our system to a pseudotyped lentivirus containing the spike of wildtype SARS-CoV-2 and the more virulent D614G, we observed progression of the infection in real time. We then found that the protease inhibitor Camostat Mesyalte significantly reduced viral transfection in distal lung epithelium. In summary, our data show that a mature human distal lung epithelium can serve as a novel moderate throughput research platform to examine viral infection and to evaluate novel therapeutics ex vivo.

Preoperative CT-guided Fiducial Marker Placement for Surgical Localization of Pulmonary Nodules.

Purpose: To assess the technical success and complication rates of CT-guided fiducial marker placement for the localization of pulmonary nodules and to assess the surgical localization failure rate. Materials and Methods: This was a single-center, retrospective analysis of consecutive patients who underwent CT-guided fiducial marker placement procedures between 2014 and 2020. End points included the technical success of the fiducial marker placement, procedural complications, and the surgical localization failure rate. A two-sample t test and a Fisher exact test were used to compare continuous and categorical variables, respectively. Multivariate logistic regression was used to identify independent risk factors for complications. Results: A total of 198 preoperative CT-guided fiducial marker placement procedures were performed in 190 patients (mean age, 64 years ± 12 [standard deviation]; 121 women) to localize 205 nodules (mean size, 10 mm ± 4; mean distance to the pleura, 10 mm ± 9). The technical success rate was 98.5% (195 of 198). There were no major complications. A total of 202 nodules were resected during 193 procedures performed 5 days ± 13 after the fiducial marker placement (range, 0-123 days). Surgical localization failure occurred in one patient (0.5%). Of the resected nodules, 146 were lung cancers, 26 nodules were metastases, two were carcinoid tumors, and 28 were benign. Conclusion: The CT-guided fiducial marker placement of pulmonary nodules was safe, effective, and resulted in a low surgical localization failure rate.Keywords: CT, Percutaneous, Thorax, Lung.