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

Label-free processing of stem cell preparations by acoustophoresis

The first bone marrow transplant in a human was performed in 1959, providing evidence that cells can be used for transplantation and treatment, revealing that healing capacities lie within the human body that ought to be understood. Since then hematopoietic stem cell transplantation has developed as a standard treatment for many cancers besides other malignancies. Much research is done to understand and utilize the properties of stem cells and their progenies for clinical application and transplantation. A routinely used valuable non-invasive information source in research and clinical applications is whole blood. However, cell processing including isolation or removal of certain components is desirable for many diagnostics, research, and transplantation applications. This thesis aimed to develop and evaluate the use of a microfluidic technology, called acoustophoresis for processing human blood and bone marrow cell preparations. Acoustophoresis utilizes the phenomenon that cells can be manipulated in an ultrasonic standing wave field in microfluidic devices. In the acoustic wave field cells experience an induced movement based on their acoustophysical properties, either to the channel center (pressure node) or towards the channel walls (pressure anti-node). These properties include size, density, and compressibility also in relation to the suspending medium. This can be utilized as biophysical biomarkers in acoustic separations.In the first two articles, it has been demonstrated that neuroblastoma tumor cells (cell line and neuroblastoma patient derived-xenograft cells) could be isolated from peripheral blood and progenitor cell products without the use of labeling antibodies, which is an advantage of acoustophoresis compared to other methods. The clinical relevance for stem cell graft processing was furthermore validated by acoustophoretic removal of transplant-contaminating tumor cells (“purging”) applicable for diagnostic, prognostic as well as potentially therapeutic purposes, with preserved high cell viability and functions. Moreover, bone marrow stromal cells (BM-MSCs) could be acoustically separated based on specific properties of primary, uncultured BM-MSCs as well as cultured MSCs. In article III, proof-of-principle evidence has been provided for the acoustic separation of functionally different subsets of cultured MSCs, which provides a first step towards better characterized and possibly enhanced cell products for cellular therapies in the future. It has further been demonstrated in article IV that primary stromal cells can be enriched from BM preparations based on their distinct biophysical properties. For clinically relevant cell processing, a stable system with relevant throughput is required. Hence, in article V a new chip holder design with an improved air-cooling unit is presented and validated, providing improved heat distribution along with stable multiplexed separation of beads and leukocyte subpopulations. This was realized at increased sample throughput of 500 µL/min and 300 µL/min, respectively. To facilitate the development of acoustofluidic cell separation applications it is crucial to obtain the properties of cell populations of interest to predict separability together with optimal experimental conditions. Therefore, the study in article VI presents a method to statistically estimate cell compressibility based on acoustophoretic separation data. Collectively, the work at hand provided valuable progress towards the validation and implementation of acoustic blood as well as stem cell processing in clinically relevant applications for cell transplantation, diagnostics, and regenerative medicine

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

A Systematic Review of Mesenchymal Epithelial Transition Factor (MET) and Its Impact in the Development and Treatment of Non-Small-Cell Lung Cancer.

Lung cancer represents the leading cause of annual cancer-related deaths worldwide, accounting for 12.9%. The available treatment options for patients who experience disease progression remain limited. Targeted therapeutic approaches are promising but further understanding of the role of genetic alterations in tumorigenesis is imperative. The MET gene has garnered great interest in this regard. The aim of this systematic review was to analyze the findings from multiple studies to provide a comprehensive and unbiased summary of the evidence. A systematic search was conducted in the reputable scientific databases Embase and PubMed, leading to the inclusion of twenty-two articles, following the PRISMA guidelines, elucidating the biological role of MET in lung cancer and targeted therapies. The systematic review was registered in PROSPERO with registration ID: CRD42023437714. MET mutations were detected in 7.6-11.0% of cases while MET gene amplification was observed in 3.9-22.0%. Six studies showed favorable treatment outcomes utilizing MET inhibitors compared to standard treatment or placebo, with increases in PFS and OS ranging from 0.9 to 12.4 and 7.2 to 24.2 months, respectively, and one study reporting an increase in ORR by 17.3%. Furthermore, patients with a higher mutational burden may derive greater benefit from treatment with MET tyrosine kinase inhibitors (TKIs) than those with a lower mutational burden. Conversely, two studies reported no beneficial effect from adjunctive treatment with a MET targeted therapy. Given these findings, there is an urgent need to identify effective therapeutic strategies specifically targeting the MET gene in lung cancer patients

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

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

Label-free separation of leukocyte subpopulations using high throughput multiplex acoustophoresis

Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 μm beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 μL min -1 corresponding to a throughput of ∼2.5 × 10 6 beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 ± 1.6%, 71.8 ± 10.1% and 98.8 ± 0.5%, respectively, as well as high efficiencies (96.8 ± 3.3%, 66.7 ± 3.2% and 99.0 ± 0.7%) at flow rates up to 100 μL min -1 (∼100000 cells per min). By increasing the flow rate up to 300 μL min -1 (∼300000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 ± 1.9%, 98.2 ± 1.0%), whereas the monocyte purity decreased to 20.9 ± 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates

Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples

Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal ("purging") from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60-97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5-2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60-80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products

Xylene versus Isopropanol for Paraffin Wax Processing of Lung Tissue

The microscopic observation of lung tissue is challenging due to its fragile nature. Xylene and isopropanol are common tissue-clearing reagents used before paraffin embedding, yet no studies have compared these two reagents in lung tissue processing. Due to the well-known health risks xylene could introduce to operators, as well as its environmental hazards, it has long been desired that a less harmful alternative to xylene with the same staining effects be introduced. Thus, we systematically assessed the efficacy of isopropanol as a substitution for xylene. Lung tissue obtained from diseased donors and explanted lungs from recipients were processed simultaneously using either xylene or isopropanol prior to paraffin embedding. Scoring of the overall staining quality after H&E staining, along with the ease of sectioning, was compared systematically. Fluorescent staining was performed to explore alveolar morphology and the overall lectin fluorescence signal between groups. To understand differences in antibody staining, the signal-to-noise ratio (SNR) of smooth muscle actin (SMA) and elastin was examined. No difference was observed with regard to ease of sectioning, staining quality, alveolar circularity, alveolar wall thickness or the SNR between slides processed with xylene or isopropanol. This study demonstrated comparable outcomes of isopropanol and xylene in lung tissue processing, suggesting isopropanol as a more favorable, operator- and environment-friendly substitute for xylene with regards to tissue processing

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use