Amniotic fluid derived mesenchymal stem cells reduce inflammation and improve lung function following transplantation in a porcine model.

BACKGROUND: Lung transplantation is hindered by low donor lung utilization rates. Infectious complications are reasons to decline donor grafts due to fear of post-transplant primary graft dysfunction. Mesenchymal stem cells are a promising therapy currently investigated in treating lung injury. Full-term amniotic fluid-derived lung-specific mesenchymal stem cell treatment may regenerate damaged lungs. These cells have previously demonstrated inflammatory mediation in other respiratory diseases, and we hypothesized that treatment would improve donor lung quality and post-operative outcomes. METHODS: In a transplantation model, donor pigs were stratified to either the treated or the non-treated group. Acute respiratory distress syndrome was induced in donor pigs and harvested lungs were placed on ex vivo lung perfusion before transplantation. Treatment consisted of three doses of 2x106 cells/kg: one during ex vivo lung perfusion and two after transplantation. Donors and recipients were assessed on clinically relevant parameters and recipients were followed for 3 days before evaluation for primary graft dysfunction (PGD). RESULTS: Repeated injection of the cell treatment showed reductions in inflammation seen through lowered immune cell counts, reduced histology signs of inflammation, and decreased cytokines in the plasma and bronchoalveolar lavage fluid. Treated recipients showed improved pulmonary function, including increased PaO2/FiO2 ratios and reduced incidence of PGD. CONCLUSIONS: Repeated injection of lung-specific cell treatment during EVLP and post-transplant was associated with improved function of previously damaged lungs. Cell treatment may be considered as a potential therapy to increase the number of lungs available for transplantation and the improvement of post-operative outcomes.

Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath.

Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7-8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7-307.4) ppm, p = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.

Proteomic changes to immune and inflammatory processes underlie lung preservation using ex vivo cytokine adsorption.

Introduction: In recent years, the field of graft preservation has made considerable strides in improving outcomes related to solid organ restoration and regeneration. Ex vivo lung perfusion (EVLP) in line with the related devices and treatments has yielded promising results within preclinical and clinical studies, with the potential to improve graft quality. Its main benefit is to render marginal and declined donor lungs suitable for transplantation, ultimately increasing the donor pool available for transplantation. In addition, using such therapies in machine perfusion could also increase preservation time, facilitating logistical planning. Cytokine adsorption has been demonstrated as a potentially safe and effective therapy when applied to the EVLP circuit and post-transplantation. However, the mechanism by which this therapy improves the donor lung on a molecular basis is not yet fully understood. Methods: We hypothesized that there were characteristic inflammatory and immunomodulatory differences between the lungs treated with and without cytokine adsorption, reflecting proteomic changes in the gene ontology pathways and across inflammation-related proteins. In this study, we investigate the molecular mechanisms and signaling pathways of how cytokine adsorption impacts lung function when used during EVLP and post-transplantation as hemoperfusion in a porcine model. Lung tissues during EVLP and post-lung transplantation were analyzed for their proteomic profiles using mass spectrometry. Results: We found through gene set enrichment analysis that the inflammatory and immune processes and coagulation pathways were significantly affected by the cytokine treatment after EVLP and transplantation. Conclusion: In conclusion, we showed that the molecular mechanisms are using a proteomic approach behind the previously reported effects of cytokine adsorption when compared to the non-treated transplant recipients undergoing EVLP.

Nothing but NETs: Cytokine adsorption correlates with lower circulating nucleosomes and is associated with decreased primary graft dysfunction.

Elevated levels of neutrophil extracellular traps (NETs) have been reported in primary graft dysfunction, making methods to reduce or remove them highly valuable. The mechanisms behind primary graft dysfunction (PGD) remain rudimentarily understood but its relation to higher rates of acute and chronic rejection necessitates the development of preventative treatments. This case series explores the use of a cytokine adsorber during lung transplantation with the focus of reducing circulating nucleosome levels as a measure of NETs. Treated patients showed reduced levels of circulating nucleosomes and remained free from PGD and histopathological signs of acute rejection at 1- and 3-month post-transplant. In contrast, patients without the adsorber experienced higher levels of circulating nucleosomes, PGD grades 1 and 3, and histopathological signs of acute rejection. Using a cytokine adsorber during transplantation may provide a reduced systemic inflammatory state with lower levels of NETs and consequently support graft acceptance.

Integrin α10ß1-selected mesenchymal stem cells reduced hypercoagulopathy in a porcine model of acute respiratory distress syndrome.

Mesenchymal stem cells (MSCs) have been studied for their potential benefits in treating acute respiratory distress syndrome (ARDS) and have reported mild effects when trialed within human clinical trials. MSCs have been investigated in preclinical models with efficacy when administered at the time of lung injury. Human integrin α10ß1-selected adipose tissue-derived MSCs (integrin α10ß1-MSCs) have shown immunomodulatory and regenerative effects in various disease models. We hypothesized that integrin α10ß1 selected-MSCs can be used to treat a sepsis-induced ARDS in a porcine model when administering cells after established injury rather than simultaneously. This was hypothesized to reflect a clinical picture of treatment with MSCs in human ARDS. 12 pigs were randomized to the treated or placebo-controlled group prior to the induction of mild to moderate ARDS via lipopolysaccharide administration. The treated group received 5 × 106 cells/kg integrin α10ß1-selected MSCs and both groups were followed for 12 h. ARDS was confirmed with blood gases and retrospectively with histological changes. After intervention, the treated group showed decreased need for inotropic support, fewer signs of histopathological lung injury including less alveolar wall thickening and reduction of the hypercoagulative disease state. The MSC treatment was not associated with adverse events over the monitoring period. This provides new opportunities to investigate integrin α10ß1-selected MSCs as a treatment for a disease which does not yet have any definitive therapeutic options.

Proteomic characteristics and diagnostic potential of exhaled breath particles in patients with COVID-19.

BACKGROUND: SARS-CoV-2 has been shown to predominantly infect the airways and the respiratory tract and too often have an unpredictable and different pathologic pattern compared to other respiratory diseases. Current clinical diagnostical tools in pulmonary medicine expose patients to harmful radiation, are too unspecific or even invasive. Proteomic analysis of exhaled breath particles (EBPs) in contrast, are non-invasive, sample directly from the pathological source and presents as a novel explorative and diagnostical tool. METHODS: Patients with PCR-verified COVID-19 infection (COV-POS, n = 20), and patients with respiratory symptoms but with > 2 negative polymerase chain reaction (PCR) tests (COV-NEG, n = 16) and healthy controls (HCO, n = 12) were prospectively recruited. EBPs were collected using a "particles in exhaled air" (PExA 2.0) device. Particle per exhaled volume (PEV) and size distribution profiles were compared. Proteins were analyzed using liquid chromatography-mass spectrometry. A random forest machine learning classification model was then trained and validated on EBP data achieving an accuracy of 0.92. RESULTS: Significant increases in PEV and changes in size distribution profiles of EBPs was seen in COV-POS and COV-NEG compared to healthy controls. We achieved a deep proteome profiling of EBP across the three groups with proteins involved in immune activation, acute phase response, cell adhesion, blood coagulation, and known components of the respiratory tract lining fluid, among others. We demonstrated promising results for the use of an integrated EBP biomarker panel together with particle concentration for diagnosis of COVID-19 as well as a robust method for protein identification in EBPs. CONCLUSION: Our results demonstrate the promising potential for the use of EBP fingerprints in biomarker discovery and for diagnosing pulmonary diseases, rapidly and non-invasively with minimal patient discomfort.

Quantitative Proteomics Indicate Radical Removal of Non-Small Cell Lung Cancer and Predict Outcome.

Non-small cell lung cancer (NSCLC) is associated with low survival rates, often due to late diagnosis and lack of personalized medicine. Diagnosing and monitoring NSCLC using blood samples has lately gained interest due to its less invasive nature. In the present study, plasma was collected at three timepoints and analyzed using proximity extension assay technology and quantitative real-time polymerase chain reaction in patients with primary NSCLC stages IA-IIIA undergoing surgery. Results were adjusted for patient demographics, tumor, node, metastasis (TNM) stage, and multiple testing. Major histocompatibility (MHC) class 1 polypeptide-related sequence A/B (MIC-A/B) and tumor necrosis factor ligand superfamily member 6 (FASLG) were significantly increased post-surgery, suggesting radical removal of cancerous cells. Levels of hepatocyte growth factor (HGF) initially increased postoperatively but were later lowered, potentially indicating radical removal of malignant cells. The levels of FASLG in patients who later died or had a relapse of NSCLC were lower at all three timepoints compared to surviving patients without relapse, indicating that FASLG may be used as a prognostic biomarker. The biomarkers were confirmed using microarray data. In conclusion, quantitative proteomics could be used for NSCLC identification but may also provide information on radical surgical removal of NSCLC and post-surgical prognosis.

Reduction of primary graft dysfunction using cytokine adsorption during organ preservation and after lung transplantation.

Despite improvements, lung transplantation remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine adsorption as a means of treating ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 16 donor pigs. Lungs were then treated with or without cytokine adsorption during ex vivo lung perfusion (EVLP) and/or post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine adsorption was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

Taking a Deep Breath: an Examination of Current Controversies in Surgical Procedures in Lung Transplantation.

Purpose of Review: This article reviews controversial questions within the field of lung transplantation, with a focus on data generated within the last 3 years. We aim to summarize differing opinions on a selection of topics, including bridge-to-transplantation, intraoperative machine circulatory support, bronchial anastomosis, size mismatch, delayed chest closure, and ex vivo lung perfusion. Recent Findings: With the growing rate of lung transplantations worldwide and increasing numbers of patients placed on waiting lists, the importance of determining best practices has only increased in recent years. Factors which promote successful outcomes have been identified across all the topics, with certain approaches promoted, such as ambulation in bridge-to-transplant and widespread intraoperative ECMO as machine support. Summary: While great strides have been made in the operative procedures involved in lung transplantation, there are still key questions to be answered. The consensus which can be reached will be instrumental in further improving outcomes in recipients.

Current Status and Future Perspectives on Machine Perfusion: A Treatment Platform to Restore and Regenerate Injured Lungs Using Cell and Cytokine Adsorption Therapy.

Since its advent in the 1990's, ex vivo lung perfusion (EVLP) has been studied and implemented as a tool to evaluate the quality of a donor organ prior to transplantation. It provides an invaluable window of opportunity for therapeutic intervention to render marginal lungs viable for transplantation. This ultimately aligns with the need of the lung transplant field to increase the number of available donor organs given critical shortages. As transplantation is the only option for patients with end-stage lung disease, advancements in technology are needed to decrease wait-list time and mortality. This review summarizes the results from the application of EVLP as a therapeutic intervention and focuses on the use of the platform with regard to cell therapies, cell product therapies, and cytokine filtration among other technologies. This review will summarize both the clinical and translational science being conducted in these aspects and will highlight the opportunities for EVLP to be developed as a powerful tool to increase the donor lung supply.