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.

High resolution fluorescence imaging of the alveolar scaffold as a novel tool to assess lung injury.

Acute lung injury (ALI) represents an aetiologically diverse form of pulmonary damage. Part of the assessment and diagnosis of ALI depends on skilled observer-based scoring of brightfield microscopy tissue sections. Although this readout is sufficient to determine gross alterations in tissue structure, its categorical scores lack the sensitivity to describe more subtle changes in lung morphology. To generate a more sensitive readout of alveolar perturbation we carried out high resolution immunofluorescence imaging on 200 µm lung vibratome sections from baseline and acutely injured porcine lung tissue, stained with a tomato lectin, Lycopersicon Esculentum Dylight-488. With the ability to resolve individual alveoli along with their inner and outer wall we generated continuous readouts of alveolar wall thickness and circularity. From 212 alveoli traced from 10 baseline lung samples we established normal distributions for alveolar wall thickness (27.37; 95% CI [26.48:28.26]) and circularity (0.8609; 95% CI [0.8482:0.8667]) in healthy tissue. Compared to acutely injured lung tissue baseline tissue exhibited a significantly lower wall thickness (26.86 ± 0.4998 vs 50.55 ± 4.468; p = 0.0003) and higher degree of circularityϕ≤ (0.8783 ± 0.01965 vs 0.4133 ± 0.04366; p < 0.0001). These two components were subsequently combined into a single more sensitive variable, termed the morphological quotient (MQ), which exhibited a significant negative correlation (R2 = 0.9919, p < 0.0001) with the gold standard of observer-based scoring. Through the utilisation of advanced light imaging we show it is possible to generate sensitive continuous datasets describing fundamental morphological changes that arise in acute lung injury. These data represent valuable new analytical tools that can be used to precisely benchmark changes in alveolar morphology both in disease/injury as well as in response to treatment/therapy.

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.

Extracorporeal Membrane Oxygenation: Set-up, Indications, and Complications.

Extracorporeal membrane oxygenation (ECMO) occupies an increasingly important position in the clinic for the management of cardiac and/or pulmonary failure. As a rescue therapy, ECMO can support patients following respiratory or cardiac compromise to act as a bridge to recovery, to decision, or to transplant. This chapter reviews briefly the history of ECMO implementation as well as device modes, from veno-arterial, veno-venous, veno-arterial-venous, and veno-venous-arterial set-ups. The importance of acknowledging complications that can arise in each of these modes cannot be overlooked. Both bleeding and thrombosis are inherent risks to the use of ECMO and the existing strategies for management are reviewed. The device also elicits an inflammatory response, and the use of extracorporeal approaches can lead to infection, both of which are important to examine when reflecting how ECMO can be successfully implemented in patients. This chapter both discusses the understanding of these various complications and highlights the need for future research.

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.

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.

Corticotropin releasing hormone as an identifier of bronchiolitis obliterans syndrome.

Lung transplantion (LTx) recipients have low long-term survival and a high incidence of bronchiolitis obliterans syndrome (BOS), an inflammation of the small airways in chronic rejection of a lung allograft. There is great clinical need for a minimally invasive biomarker of BOS. Here, 644 different proteins were analyzed to detect biomarkers that distinguish BOS grade 0 from grades 1-3. The plasma of 46 double lung transplant patients was analyzed for proteins using a high-component, multiplex immunoassay that enables analysis of protein biomarkers. Proximity Extension Assay (PEA) consists of antibody probe pairs which bind to targets. The resulting polymerase chain reaction (PCR) reporter sequence can be quantified by real-time PCR. Samples were collected at baseline and 1-year post transplantation. Enzyme-linked immunosorbent assay (ELISA) was used to validate the findings of the PEA analysis across both time points and microarray datasets from other lung transplantation centers demonstrated the same findings. Significant decreases in the plasma protein levels of CRH, FERC2, IL-20RA, TNFB, and IGSF3 and an increase in MMP-9 and CTSL1 were seen in patients who developed BOS compared to those who did not. In this study, CRH is presented as a novel potential biomarker in the progression of disease because of its decreased levels in patients across all BOS grades. Additionally, biomarkers involving the remodeling of the extracellular matrix (ECM), such as MMP-9 and CTSL1, were increased in BOS patients.

The role of mechanical ventilation in primary graft dysfunction in the postoperative lung transplant recipient: A single center study and literature review.

BACKGROUND: Primary graft dysfunction (PGD) is still a major complication in patients undergoing lung transplantation (LTx). Much is unknown about the effect of postoperative mechanical ventilation on outcomes, with debate on the best approach to ventilation. AIM/PURPOSE: The goal of this study was to generate hypotheses on the association between postoperative mechanical ventilation settings and allograft size matching in PGD development. METHOD: This is a retrospective study of LTx patients between September 2011 and September 2018 (n = 116). PGD was assessed according to the International Society of Heart and Lung Transplantation (ISHLT) criteria. Data were collected from medical records, including chest x-ray assessments, blood gas analysis, mechanical ventilator parameters and spirometry. RESULTS: Positive end-expiratory pressures (PEEP) of 5 cm H2 O were correlated with lower rates of grade 3 PGD. Graft size was important as tidal volumes calculated according to the recipient yielded greater rates of PGD when low volumes were used, a correlation that was lost when donor metrics were used. CONCLUSION: Our results highlight a need for greater investigation of the role donor characteristics play in determining post-operative ventilation of a lung transplant recipient. The mechanical ventilation settings on postoperative LTx recipients may have an implication for the development of acute graft dysfunction. Severe PGD was associated with the use of a PEEP higher than 5 and lower tidal volumes and oversized lungs were associated with lower long-term mortality. Lack of association between ventilatory settings and survival may point to the importance of other variables than ventilation in the development of PGD.

Monitoring lung injury with particle flow rate in LPS- and COVID-19-induced ARDS.

In severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) is a life-prolonging treatment, especially among COVID-19 patients. Evaluation of lung injury progression is challenging with current techniques. Diagnostic imaging or invasive diagnostics are risky given the difficulties of intra-hospital transportation, contraindication of biopsies, and the potential for the spread of infections, such as in COVID-19 patients. We have recently shown that particle flow rate (PFR) from exhaled breath could be a noninvasive, early detection method for ARDS during mechanical ventilation. We hypothesized that PFR could also measure the progress of lung injury during ECMO treatment. Lipopolysaccharide (LPS) was thus used to induce ARDS in pigs under mechanical ventilation. Eight were connected to ECMO, whereas seven animals were not. In addition, six animals received sham treatment with saline. Four human patients with ECMO and ARDS were also monitored. In the pigs, as lung injury ensued, the PFR dramatically increased and a particular spike followed the establishment of ECMO in the LPS-treated animals. PFR remained elevated in all animals with no signs of lung recovery. In the human patients, in the two that recovered, PFR decreased. In the two whose lung function deteriorated while on ECMO, there was increased PFR with no sign of recovery in lung function. The present results indicate that real-time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS.

Age is But a Number: Damage Control Surgery Outcomes in Geriatric Emergency General Surgery.

BACKGROUND: Damage control surgery (DCS) with temporary abdominal closure (TAC) is increasingly utilized in emergency general surgery (EGS). As the population ages, more geriatric patients (GP) are undergoing EGS operations. Concern exists for GP's ability to tolerate DCS. We hypothesize that DCS in GP does not increase morbidity or mortality and has similar rates of primary closure compared to non-geriatric patients (NGP). METHODS: A retrospective chart review from 2014-2020 was conducted on all non-trauma EGS patients who underwent DCS with TAC. Demographics, admission lab values, fluid amounts, length of stay (LOS), timing of closure, post-operative complications and mortality were collected. GP were compared to NGP and results were analyzed using Chi square and Wilcox signed rank test. RESULTS: Ninety-eight patients (n = 50, <65 y; n = 48, ≥65 y) met inclusion criteria. There was no significant difference in median number of operations (3 versus 2), time to primary closure (2.5 versus 3 d), hospital LOS (19 versus 17.5 d), ICU LOS (11 versus 8 d), rate of primary closure (66% versus 56%), post op ileus (44% versus 48%), abscess (14% versus 10%), need for surgery after closure (32% versus 19%), anastomotic dehiscence (16% versus 6%), or mortality (34% versus 42%). Average time until take back after index procedure did not vary significantly between young and elderly group (45.8 versus 38.5 h; P = 0.89). GP were more likely to have hypertension (83% versus 50%; P ≤ 0.05), atrial fibrillation (25% versus 4%; P ≤ 0.05) and lower median heart rate compared to NGP (90 versus 103; P ≤ 0.05). CONCLUSIONS: DCS with TAC in geriatric EGS patients achieves similar outcomes and mortality to younger patients. Indication, not age, should factor into the decision to perform DCS.

Particle flow rate from the airways as fingerprint diagnostics in mechanical ventilation in the intensive care unit: a randomised controlled study.

INTRODUCTION: Mechanical ventilation can be monitored by analysing particles in exhaled air as measured by particle flow rate (PFR). This could be a potential method of detecting ventilator-induced lung injury (VILI) before changes in conventional parameters can be detected. The aim of this study was to investigate PFR during different ventilation modes in patients without lung pathology. METHOD: A prospective study was conducted on patients on mechanical ventilation in the cardiothoracic intensive care unit (ICU). A PExA 2.0 device was connected to the expiratory limb on the ventilator for continuous measurement of PFR in 30 patients randomised to either volume-controlled ventilation (VCV) or pressure-controlled ventilation (PCV) for 30 min including a recruitment manoeuvre. PFR measurements were continued as the patients were transitioned to pressure-regulated volume control (PRVC) and then pressure support ventilation (PSV) until extubation. RESULTS: PRVC resulted in significantly lower PFR, while those on PSV had the highest PFR. The distribution of particles differed significantly between the different ventilation modes. CONCLUSIONS: Measuring PFR is safe after cardiac surgery in the ICU and may constitute a novel method of continuously monitoring the small airways in real time. A low PFR during mechanical ventilation may correlate to a gentle ventilation strategy. PFR increases as the patient transitions from controlled mechanical ventilation to autonomous breathing, which most likely occurs as recruitment by the diaphragm opens up more distal airways. Different ventilation modes resulted in unique particle patterns and could be used as a fingerprint for the different ventilation modes.

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.

Isolation of high-yield and -quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts.

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.