Pig Lung Research Articles

Negative Pressure Ventilation Ex-Situ Lung Perfusion Preserves Porcine and Human Lungs for 36-Hours.

Preclinically, 24-hour continuous Ex-Situ Lung Perfusion (ESLP) is the longest duration achieved in large animal models and rejected human lungs. Here, we present our 36-hour Negative Pressure Ventilation (NPV)-ESLP protocol applied to porcine and rejected human lungs. Five sets of donor domestic pig lungs (45-55kg) underwent 36-hour NPV-ESLP. Two sets of clinically rejected human lungs were preserved on 36-hour NPV-ESLP. Graft function was assessed via physiologic parameters, edema formation, and cytokine profiles. Porcine and human lung function was stable with mean partial pressure of oxygen divided by the fraction of inspired oxygen (PaO2/FiO2; PF) ratios throughout preservation of 473±11.79 and 554.7±13.26, respectively (mean±standard error of the mean). In porcine lungs, mean compliance (Cdyn) during ESLP was 33.96±2.18, pulmonary artery pressure (PAP) 13.03±0.53, and pulmonary vascular resistance (PVR) 481.20 ±21.86. In human lungs, mean Cdyn was 82.68±3.54, PAP 6.00±0.33, and PVR 184.00±9.71. Average percentage weight-gain was 34.47±13.22 in porcine lungs and 116.3±6.65 in rejected human lungs. NPV-ESLP can preserve porcine lungs and human lungs for 36-hours with acceptable physiologic function. Greater weight-gain in the human lungs is likely due to prolonged ischemic time prior to ESLP and use of an acellular perfusate. Continuous 36-hour NPV-ESLP could support therapies for endothelial protection and mitigate fluid accumulation.

Feasibility of computed tomography-derived surgical margin assessment in an ex vivo sublobar lung resection model.

Computed tomography imaging of a sublobar resection specimen may inform intraoperative surgical margin assessment. However, consistency with final pathological margins has not been previously evaluated. In this study, we investigated the concordance between surgical margin measurements by computed tomography versus pathology measurements using an ex vivo sublobar lung resection model. Pig lung wedge samples containing agarose pseudotumors were harvested. Computed tomography images were acquired following specimen inflation. The specimen was bisected along the same plane observed by computed tomography for accurate comparison with pathological surgical margin measurement. The bisected samples were then fixed in formalin before preparing hematoxylin & eosin slides. Surgical margin length at four distinct stages (computed tomography, gross pre-formalin fixation, gross post-formalin fixation, pathology) were measured and compared. A total of 50 lung specimens were analyzed. After specimen processing, Surgical margin length decreased in 94% (47/50) and increased in 6% (3/50) of samples. Mean surgical margin lengths were as follows: computed tomography 14.0 mm (range: 4.5-28.3 mm), gross pre-formalin fixation 13.0 mm (range: 4.0-25.0 mm), gross post-formalin fixation 12.1 mm (range: 2.5-26.0 mm), and pathology 10.9 mm (range: 1.0-23.4 mm). There was an average -23.8% (range: +11 to -82%) change in surgical margin length from computed tomography to final pathology (p < 0.001). While computed tomography -based surgical margin measurement is feasible, we observed an average 23.8% discordance when compared to final pathology measurement. Surgeons must be aware that the computed tomography -derived surgical margin generally overestimates the pathology-derived surgical margin.

Phenotypic and Genotypic Analysis of Antimicrobial Resistance in Mycoplasma hyopneumoniae Isolated from Pigs with Enzootic Pneumonia in Australia.

Mycoplasma hyopneumoniae, an important cause of enzootic pneumonia in pigs in many countries, has recently been shown to exhibit reduced susceptibility to several antimicrobial classes. In the present study, a total of 185 pig lung tissue samples were collected from abattoirs in Australia, from which 21 isolates of M. hyopneumoniae were obtained. The antimicrobial resistance profile of the isolates was determined for 12 antimicrobials using minimum inhibitory concentration (MIC) testing, and a subset (n = 14) underwent whole-genome sequence analysis. MIC testing revealed uniformly low values for enrofloxacin (≤1 μg/mL), florfenicol (≤8 μg/mL), lincomycin (≤4 μg/mL), spectinomycin (≤4 μg/mL), tetracycline (≤0.5 μg/mL), tiamulin (≤2 μg/mL), tildipirosin (≤4 μg/mL), tilmicosin (≤16 μg/mL) tulathromycin (≤2 μg/mL), and tylosin (≤2 μg/mL). Higher MICs were observed for erythromycin (MIC range: 16-32 μg/mL), gamithromycin, and tilmicosin (MIC range of both: 32-64 μg/mL). Whole-genome sequencing of the isolates and additional screening using mismatch amplification mutation assay PCR did not identify any known genetic resistance markers within 23S rRNA (macrolides), DNA gyrase A, and topoisomerase IV genes (fluoroquinolones). The WGS data also indicated that the Australian M. hyopneumoniae isolates exhibited limited genetic diversity and formed a distinct monophylectic clade when compared to isolates from other countries. These findings indicate that Australian M. hyopneumoniae likely remains susceptible to the major antimicrobials used to treat enzootic pneumonia in pigs and have evolved in isolation from strains identified in other pig-producing countries.

Mild Permissive Alkalosis Improves Outcomes in Porcine Negative Pressure Ventilation Ex-Situ Lung Perfusion

Ex-Situ Lung Perfusion (ESLP) employs a membrane deoxygenator and mixed (N2/O2/CO2) or pure sweep gas (CO2) to target venous blood gas composition with physiologic pCO2 and pH. Clinically, mild permissive alkalosis counteracts elevated pulmonary vascular resistance (PVR) to improve perfusion. Increased PVR and pulmonary artery pressure (PAP) during ESLP mirrors rising pro-inflammatory cytokines. Increased hydrostatic pressure worsens edema and lung function. We report improved ESLP outcomes using mild permissive alkalosis. Twelve juvenile pig lungs underwent 12-hour Negative Pressure Ventilation (NPV)-ESLP with a physiologic pH (Control: pH 7.35-7.45, n=6) or mild permissive alkalosis (pH+: pH 7.45-7.55, n=6) by varying sweep CO2 delivery. Three left lungs per group were transplanted and assessed over 4-hours. Five Control lungs failed on ESLP due to high PAPs, low compliance, and poor oxygenation. Repeat Controls (n=6) were performed to attain 12-hours of ESLP. There were no failures in the pH+ group. Results are pH+ vs Control. Oxygenation (PaO2/FiO2 454.2 vs 438.2; P = .37) and dynamic compliance (21.38 vs 22.22 mL/cmH2O; P = .41) were stable over 12-hour NPV-ESLP. Mean evaluation pH/pCO2/HCO3- was 7.50/15.6/14.5 vs 7.41/38.7/24.7. Control lungs required repeat THAM and milrinone boluses on ESLP to prevent acidosis and treat elevated PVR; this was not necessary in the pH+ group. Weight-gain/hour was similar (1.23% vs 1.38%; P = .37). Mean left lung PF ratios 4-hours post-transplantation were 301 mmHg vs 196 mmHg (P = .11). Control TNF-⍺ and IL-6 perfusate concentrations were significantly greater. Mild permissive alkalosis porcine NPV-ESLP demonstrated more reliable preservation with reduced inflammation compared to a physiologic pH strategy.

Development of a Desorption Electrospray Ionization-Multiple-Reaction-Monitoring Mass Spectrometry (DESI-MRM) Workflow for Spatially Mapping Oxylipins in Pulmonary Tissue.

Oxylipins are a class of low-abundance lipids formed via oxygenation of fatty acids. These compounds include potent signaling molecules (e.g., octadecanoids, eicosanoids) that can exert essential functions in the pathophysiology of inflammatory diseases including asthma. While some oxylipin signaling cascades have been unraveled using LC-MS/MS-based methods, measurements in homogenate samples do not represent the spatial heterogeneity of lipid metabolism. Mass spectrometry imaging (MSI) directly detects analytes from a surface, which enables spatial mapping of oxylipin biosynthesis and migration within the tissue. MSI has lacked the sensitivity to routinely detect low-abundance oxylipins; however, new multiple-reaction-monitoring (MRM)-based MSI technologies show increased sensitivity. In this study, we developed a workflow to apply desorption electrospray ionization coupled to a triple quadrupole mass spectrometer (DESI-MRM) to spatially map oxylipins in pulmonary tissue. The targeted MSI workflow screened guinea pig lung extracts using LC-MS/MS to filter oxylipin targets based on their detectability by DESI-MRM. A panel of 5 oxylipins was then selected for DESI-MRM imaging derived from arachidonic acid (TXB2, 11-HETE, 12-HETE), linoleic acid (12,13-DiHOME), and α-linolenic acid (16-HOTrE). To parse this new data type, a custom-built R package (quantMSImageR) was developed with functionality to label regions of interest as well as quantify and analyze lipid distributions. The spatial distributions quantified by DESI-MRM were supported by LC-MS/MS analysis, with both indicating that 16-HOTrE and 12-HETE were associated with airways, while 12,13-DiHOME and arachidonic acid were mapped to parenchyma. This study realizes the potential of targeted MSI to routinely map low-abundance oxylipins with high specificity at scale.

3D Bioprinting of Pig Macrophages and Human Cells Discovered the P2Y14 Receptor as a Mediator of Xenogenic Immune Responses

ABSTRACT Background The survival rate of pig lung xenotransplantation (PLXTx) recipients is severely limited by intense xenogenic immune responses, necessitating further insights into xenogeneic immunity and the development of models to study the PLXTx immune response. Methods We identified regulators of PLXTx immune response Using Gene ontology analysis. We assessed the metabolic changes and protein levels in 3D4/31 pig alveolar macrophages (PAMs) through flow cytometry and immunoblotting. To induce a xenogenic immune response, we co-cultured 3D4/31-PAMs with A549 human alveolar epithelial cells and evaluated cytokine expression using qRT-PCR. Results Gene ontology analysis identified STAT1 and alveolar macrophages as contributors to lung autoimmunity and transplant rejection. In 3D4/31-PAMs, phorbol myristate acetate-induced glycogen accumulation and cyclooxygenase-2 expression were inhibited by the P2Y14 inhibitor PPTN. Co-culturing 3D4/31-PAMs with A549 human alveolar epithelial cells via 3D bioprinting resulted in a more pronounced inflammatory response than 2D co-culture, with increased expression of genes related to the P2Y14 cascade and inflammation. This inflammatory gene expression was prevented by PPTN treatment. Conclusion Based on these results, we propose alginate bioprinting as an in vitro model for PLXTx and suggest that P2Y14 is a key regulator of xenogeneic immune responses in PAMs.

Structural and quantitative comparison of viral infection-associated N-glycans in plasma from humans, pigs, and chickens: Greater similarity between humans and chickens than pigs

Host N-glycans play an essential role in the attachment, invasion, and infection processes of viruses, including zoonotic infectious diseases. The similarity of N-glycans in the trachea and lungs of humans and pigs facilitates the cross-species transmission of influenza viruses through respiratory tracts. In this study, the structure and quantity of N-glycans in the plasma of humans, pigs, and chickens were analyzed using liquid chromatography-quadrupole-Orbitrap-tandem mass spectrometry. N-glycans in humans (35), pigs (28), and chickens (53) were identified, including the most abundant, species-common, and species-specific N-glycans. Among the N-glycans (relative quantity >0.5%), the sialic acid derivative of N-acetylneuraminic acid was identified in humans (the sum of the relative quantities of each; 64.3%), pigs (45.5%), and chickens (64.4%), whereas N-glycolylneuraminic acid was only identified in pigs (18.1%). Sialylated N-glycan linkage isomers are the influenza virus receptors (α2-6 in humans, α2-3 and α2-6 in pigs, and α2-3 in chickens). Only α2-6 linkages (human, 58.2%; pig, 44.8%; and chicken, 60.6%) were more abundant than α2-3/α2-6 linkages (human, 4.6%; pig, 0.6%; and chicken, 3.4%) and only α2-3 linkages (human, 1.5%; pig, 0.1%; and chicken, 0.4%). Fucosylation, which can promote viral infection through immune modulation, was more abundant in pigs (76.1%) than in humans (36.4%) and chickens (16.7%). Bisecting N-acetylglucosamine, which can suppress viral infection by inhibiting sialylation, was identified in humans (10.3%) and chickens (16.9%), but not in pigs. These results indicate that plasma N-glycans are similar in humans and chickens. This is the first study to compare plasma N-glycans in humans, pigs, and chickens.

Thioredoxin C of Streptococcus suis serotype 2 contributes to virulence by inducing antioxidative stress and inhibiting autophagy via the MSR1/PI3K-Akt-mTOR pathway in macrophages

The thioredoxin (Trx) system plays a vital role in protecting against oxidative stress and ensures correct disulfide bonding to maintain protein function. Our previous research demonstrated that TrxA of Streptococcus suis Serotype 2 (SS2), a clinical strain from the lung of a diseased pig, contributes to virulence but is not involved in antioxidative stress. In this study, we identified another gene in the Trx family, TrxC, which encodes a protein of 104 amino acids with a CGDC active motif and 22.4 % amino acid sequence homology with TrxA. Unlike the TrxA, TrxC mutant strains were more susceptible to oxidative stresses induced by hydrogen peroxide and paraquat. In vitro experiments, the survival rate of the TrxC deletion mutant in RAW264.7 macrophages was only one-eighth of that of TrxA mutant strains. Transcriptome analysis revealed that autophagy-related genes were significantly upregulated in the TrxC mutant compared to those in the wild-type or TrxA mutant strains. Co-localization of LC3 puncta with TrxC was confirmed using laser confocal microscopy, and autophagy-related indicators were quantified using western blotting. Autophagy deficiency induced by ATG5 knockout significantly increased SS2 survival rate, especially in TrxC mutant strains. For the upstream signal regulation pathways, we found ΔTrxC strains regulate autophagy by activation of PI3K/Akt/mTOR signaling in RAW264.7 macrophages. In the Akt1-overexpressing cell line, ΔTrxC infection significantly decreased the autophagic response and promoted ΔTrxC mutant strain survival, while inhibition of Akt with MK2206 resulted in reduced ΔTrxC mutant strain survival and enhance the autophagic response. Furthermore, loss of TrxC increased the activity of MSR1, thereby inducing cellular autophagy and phagocytosis. Our data demonstrate that TrxC of SS2 contributes to virulence by inducing antioxidative stress and inhibits autophagy via the PI3K-Akt-mTOR pathway in macrophages, with MSR1 acting as a key factor in controlling infection.

Development and method validation of a sampling technique for a reproducible detection of synthetic cannabinoids in exhaled breath using an in vitro pig lung model.

Alternative matrices, especially exhaled breath (EB), have gained increasing attention for a few years. To interpret toxicological findings, knowledge on the toxicokinetic (TK) properties of a substance in EB is indispensable. While such data are already accessible for various drugs (e.g. Δ9-tetrahydrocannabinol), they are still not available for new psychoactive substances, particularly synthetic cannabinoids (SCs). As SCs raise a high public health concern, the aim of this study was to assess these data in future TK studies in pigs. For this purpose, an in vitro sampling technique of EB was initially developed, which is prospectively applied to anesthetized and ventilated pigs for the detection of SCs in a controlled and reproducible manner as exemplified by cumyl-5F-P7AICA. Furthermore, a method for the qualitative and quantitative detection of cumyl-5F-P7AICA in EB using glass fiber filters (GFFs) was established and fully validated. Therefore, cumyl-5F-P7AICA (0.5 mg/mL in ethanol absolute) was initially nebulized using a ventilation machine and a breathing tube, as they are also used in surgeries. The aerosol was delivered into a simulated pig lung. To collect EB, a pump was connected to that part of the breathing tube, which contains EB (expiratory limb), and sampling was performed repeatedly (n = 6) for 15 min (2 l EB/min) each using GFF. For extraction of the substance, the GFFs were macerated with acetone and the remaining experimental components were rinsed with ethanol. After sample preparation, the extracts were analyzed by liquid chromatography tandem mass spectrometry. In the complete experimental setup, about 40% of the initially nebulized cumyl-5F-P7AICA dose was found, with 3.6 ± 1.3% being detected in the GFF. Regarding the comparably high loss of substance, the open ventilation system and a conceivable adsorption of the SC in the ventilator have to be considered. However, the herein introduced approach is promising to determine the TK properties of cumyl-5F-P7AICA in EB.

Visceral pleura mechanics: Characterization of human, pig, and rat lung material properties

Pulmonary air leaks are amongst the most common complications in lung surgery. Lung sealants are applied to the organ surface and need to synchronously stretch with the visceral pleura, the layer of tissue which encompasses the lung parenchymal tissue. These adhesives are commonly tested on pig and rat lungs, but applied to human lungs. However, the unknown mechanics of human lung visceral pleura undermines the clinical translatability of such animal-tested sealants and the absence of how pig and rat lung visceral pleura compare to human tissues is necessary to address. Here we quantify the biaxial planar tensile mechanics of visceral pleura from healthy transplant-eligible and smoker human lungs for the first time, and further compare the material behaviors to pig and rat lung visceral pleura. Initial and final stiffness moduli, maximum stress, low-to-high strain transition, and stress relaxation are analyzed and compared between and within groups, further considering regional and directional dependencies. Visceral pleura tissue from all species behave isotropically, and pig and human visceral pleura exhibits regional heterogeneity (i.e. upper versus lower lobe differences). We find that pig visceral pleura exhibits similar initial stiffness moduli and regional trends compared to human visceral pleura, suggesting pig tissue may serve as a viable animal model candidate for lung sealant testing. The outcomes and mechanical characterization of these scarce tissues enables future development of biomimetic lung sealants for improved surgical applications. Statement of significanceSurgical lung sealants must synchronously deform with the underlying tissue and with each breath to minimize post-operative air leaks, which remain the most frequent complications of pulmonary intervention. These adhesives are often tested on pig and rat lungs, but applied to humans; however, the material properties of human lung visceral pleura were previously unexplored. Here, for the first time, the mechanics of human visceral pleura tissue are investigated, further contrasting rarely acquired donated lungs from healthy and smoking individuals, and additionally, comparing biaxial planar material characterizations to animal models often employed for pulmonary sealant development. This fundamental material characterization addresses key hindrances in the advancement of biomimetic sealants and evaluates the translatability of animal model experiments for clinical applications.

V-312.3: Insights from platelet and leukocyte sequestration in pig lung perfusion with human blood: role of selectins, porcine sialoadhesin, and CD11b/CD18 Integrin.

V-312.3: Insights from platelet and leukocyte sequestration in pig lung perfusion with human blood: role of selectins, porcine sialoadhesin, and CD11b/CD18 Integrin.

Assessing the antiviral activity of antimicrobial peptides Caerin1.1 against PRRSV in Vitro and in Vivo

The Porcine reproductive and respiratory syndrome (PRRS) causes severe financial losses to the global swine industry. Due to continuous virus evolution, the protection against the PRRS provided by current vaccines is limited. In order to find new antiviral strategies, this study investigated the antiviral potential of antimicrobial peptides (AMPs) against PRRSV. Given the diversity of PRRSV strains and the limited effectiveness of existing vaccines in controlling PRRSV, this study evaluated the inhibitory effects of KLAK, Cecropin B, Piscidin1, and Caerin1.1 on 3 strains of PRRSV (lineage 5 classical strain, lineage 8 highly pathogenic strain, and lineage 1 NADC30-like strain). Caerin1.1 exhibited significant dose-dependent antiviral activity, with an effective concentration (EC50) of 7.5 μM. Caerin1.1 effectively inhibited PRRSV replication when added before or in early infection but showed reduced effectiveness when added in late infection, indicating its potential involvement in targeting early transcription mechanisms of viral RNA polymerase and significantly upregulating cytokine gene expression. In the NADC30 strain-based animal infection model, Caerin1.1 treatment significantly reduced lung viral loads and inflammation in the lungs of PRRSV-infected pigs, with a mortality rate of 0 % (0/5) in the treated group compared to 66.67 % (4/6) in the untreated group, indicating a reduction in the mortality rate. Additionally, compared with the untreated group, the Caerin1.1-treated group showed significant improvements, such as lighter fever, more daily weight gain, less clinical symptoms, less viral load in blood, and less virus oral shedding (P < 0.05). These findings reveal the potential of antimicrobial peptides as PRRSV therapeutic agents and suggest that Caerin1.1 is a promising candidate for a novel anti-PRRSV drug.

Endobronchial Closure for Peripheral Pulmonary Air Leakage

Introduction: A minimally invasive alternative to surgery for treating pneumothorax has been developed, aiming to reduce risks while maintaining efficacy. This study conducted basic experiments using ex vivo and in vivo pig lung employing a super-thin catheter for treatment. This new device injects fibrin glue directly into the responsible lesion to close the air leak, which has two features: thin design and double-lumen. Methods: The experimental setup involved utilizing trachea and both lung specimens from pigs under positive pressure ventilation. To simulate pneumothorax, artificial fistulas were created on the lung surfaces. The super-thin catheter, guided through a bronchoscope near the fistula, was used to embolize the peripheral bronchus by injecting a fibrin preparation. Then, an air leak test was conducted afterward to assess the efficacy of the treatment. Additionally, a similar pneumothorax model was induced in alive pig under general anesthesia to evaluate its curability. Results: In the extracted pig lungs, embolization was performed in 21 cases, resulting in the cessation of air leaks in 19 cases, corresponding to a 90.5% cure rate. Notably, no major adverse events occurred with the treatment devices. Similarly, in living pigs, pneumothorax was successfully treated, with no recurrence observed up to the seventh postoperative day. Conclusion: The novel treatment device utilizing a super-thin catheter offers a minimally invasive and highly curative option for pneumothorax. These promising results suggest the potential for further development and human clinical trials, which could revolutionize the treatment of pneumothorax, reducing risks and improving outcomes.

Developing Hyperpolarized Butane Gas for Ventilation Lung Imaging.

NMR hyperpolarization dramatically improves the detection sensitivity of magnetic resonance through the increase in nuclear spin polarization. Because of the sensitivity increase by several orders of magnitude, additional applications have been unlocked, including imaging of gases in physiologically relevant conditions. Hyperpolarized 129Xe gas recently received FDA approval as the first inhalable gaseous MRI contrast agent for clinical functional lung imaging of a wide range of pulmonary diseases. However, production and utilization of hyperpolarized 129Xe gas faces a number of translational challenges including the high cost and complexity of contrast agent production and imaging using proton-only (i.e., conventional) clinical MRI scanners, which are typically not suited to scan 129Xe nuclei. As a solution to circumvent the translational challenges of hyperpolarized 129Xe, we have recently demonstrated the feasibility of a simple and cheap process for production of proton-hyperpolarized propane gas contrast agent using ultralow-cost disposable production equipment and demonstrated the feasibility of lung ventilation imaging using hyperpolarized propane gas in excised pig lungs. However, previous pilot studies have concluded that the hyperpolarized state of propane gas decays very fast with an exponential decay T 1 constant of ∼0.8 s at 1 bar (physiologically relevant pressure); moreover, the previously reported production rates were too slow for potential clinical utilization. Here, we investigate the feasibility of high-capacity production of hyperpolarized butane gas via heterogeneous parahydrogen-induced polarization using Rh nanoparticle-based catalyst utilizing butene gas as a precursor for parahydrogen pairwise addition. We demonstrate a remarkable result: the lifetime of the hyperpolarized state can be nearly doubled compared to that of propane (T 1 of ∼1.6 s and long-lived spin-state T S of ∼3.8 s at clinically relevant 1 bar pressure). Moreover, we demonstrate a production speed of up to 0.7 standard liters of hyperpolarized gas per second. These two synergistic developments pave the way to biomedical utilization of proton-hyperpolarized gas media for ventilation imaging. Indeed, here we demonstrate the feasibility of phantom imaging of hyperpolarized butane gas in Tedlar bags and also the feasibility of subsecond 2D ventilation gas imaging in excised rabbit lungs with 1.6 × 1.6 mm2 in-plane resolution using a clinical MRI scanner. The demonstrated results have the potential to revolutionize functional pulmonary imaging with a simple and inexpensive on-demand production of proton-hyperpolarized gas contrast media, followed by visualization on virtually any MRI scanner, including emerging bedside low-field MRI scanner technology.

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunization in the pig lung.

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1β. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1β reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.

Future directions for xenotransplantation in lungs.

Advancements in preclinical xenotransplant studies have opened doors for clinical heart and kidney xenotransplantation. This review assesses recent progress in lung xenotransplantation research and its potential clinical implications. The efficacy of the humanized von Willebrand factor in reducing platelet sequestration in ex-vivo and in-vivo lung xenotransplant models was showcased. Combining human tissue factor pathway inhibitor and CD47 expression with selectin and integrin inhibition delayed neutrophil and platelet sequestration. Enhanced expression of human complement regulatory proteins and thrombomodulin in genetically engineered pig lungs improved graft survival by reducing platelet activation and modulating coagulation disruptions. Knocking out the CMAH gene decreased antibody-mediated inflammation and coagulation activation, enhancing compatibility for human transplantation. Furthermore, CMAH gene knockout in pigs attenuated sialoadhesin-dependent binding of human erythrocytes to porcine macrophages, mitigating erythrocyte sequestration and anemia. Meanwhile, in-vivo experiments demonstrated extended survival of xenografts for up to 31 days with multiple genetic modifications and comprehensive treatment strategies. Experiments have uncovered vital insights for successful xenotransplantation, driving further research into immunosuppressive therapy and genetically modified pigs. This will ultimately pave the way for clinical trials designed to improve outcomes for patients with end-stage lung disease.

Normothermic Perfusion is Superior to Cold Perfusion in Porcine Ex Situ Lung Perfusion

BackgroundCold ex situ lung perfusion (ESLP) has demonstrated improved preservation in small animal ESLP compared to normothermic ESLP and cold static preservation. We hypothesized that cold negative pressure ventilation (NPV)-ESLP would improve graft function in a porcine transplantation model. MethodsFour perfusate temperatures were examined with 12 hours NPV-ESLP in a large animal transplantation model. Pig lungs were allotted to four groups: (1) Normothermia (38°C, n = 6); (2) profound hypothermia (10°C, n = 6); (3) moderate hypothermia (20°C, n = 3); (4) subnormothermia (32°C, n = 3). A fifth group subnormothermic low-flow (SNLF) perfusion was examined to assess the effect of reduced cardiac output with cold perfusion (32°C, 10% cardiac output, n = 6). ResultsOnly Normothermic and SNLF groups demonstrated acceptable oxygenation after 12 hours NPV-ESLP and were transplanted. All other groups failed prematurely. After 12 hours of ESLP, Normothermic lungs demonstrated significantly greater dynamic compliance compared to SNLF lungs (P = .03). Edema formation post-ESLP was significantly worse in the SNLF group (P = .01). There was no significant difference in pulmonary artery pressures after ESLP (P = .10); however, pulmonary vascular resistance was significantly greater in the SNLF (P = .04). Isolated left lung oxygenation 4-hours post-transplant and left lung edema formation was not significantly different between Normothermic and SNLF post-transplant (P = .09). Proinflammatory cytokines were significantly greater during SNLF-ESLP (tumor necrosis factor alpha, P < .05). ConclusionsProlonged normothermic (38°C) NPV-ESLP is superior to 10, 20, and 32°C perfusion. Normothermic ESLP of porcine lungs results in superior graft function and reduced inflammation versus SNLF-ESLP.

Multi-omics analysis of diabetic pig lungs reveals molecular derangements underlying pulmonary complications of diabetes mellitus.

Growing evidence shows that the lung is an organ prone to injury by diabetes mellitus. However, the molecular mechanisms of these pulmonary complications have not yet been characterized comprehensively. To systematically study the effects of insulin deficiency and hyperglycaemia on the lung, we combined proteomics and lipidomics with quantitative histomorphological analyses to compare lung tissue samples from a clinically relevant pig model for mutant INS gene-induced diabetes of youth (MIDY) with samples from wild-type littermate controls. Among others, the level of pulmonary surfactant-associated protein A (SFTPA1), a biomarker of lung injury, was moderately elevated. Furthermore, key proteins related to humoral immune response and extracellular matrix organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by 2.5-fold reduction of polyunsaturated fatty acid lipoxygenase ALOX15 levels, associated with corresponding changes in the levels of lipids influenced by this enzyme. Our multi-omics study points to an involvement of reduced ALOX15 levels and an associated lack of eicosanoid switching as mechanisms contributing to a proinflammatory milieu in the lungs of subjects with diabetes mellitus.

Recreating chronic respiratory infections in vitro using physiologically relevant models.

Despite the need for effective treatments against chronic respiratory infections (often caused by pathogenic biofilms), only a few new antimicrobials have been introduced to the market in recent decades. Although different factors impede the successful advancement of antimicrobial candidates from the bench to the clinic, a major driver is the use of poorly predictive model systems in preclinical research. To bridge this translational gap, significant efforts have been made to develop physiologically relevant models capable of recapitulating the key aspects of the airway microenvironment that are known to influence infection dynamics and antimicrobial activity in vivo In this review, we provide an overview of state-of-the-art cell culture platforms and ex vivo models that have been used to model chronic (biofilm-associated) airway infections, including air-liquid interfaces, three-dimensional cultures obtained with rotating-wall vessel bioreactors, lung-on-a-chips and ex vivo pig lungs. Our focus is on highlighting the advantages of these infection models over standard (abiotic) biofilm methods by describing studies that have benefited from these platforms to investigate chronic bacterial infections and explore novel antibiofilm strategies. Furthermore, we discuss the challenges that still need to be overcome to ensure the widespread application of in vivo-like infection models in antimicrobial drug development, suggesting possible directions for future research. Bearing in mind that no single model is able to faithfully capture the full complexity of the (infected) airways, we emphasise the importance of informed model selection in order to generate clinically relevant experimental data.

Glycyrrhizin alleviates BoAHV-1-induced lung injury in guinea pigs by inhibiting the NF-κB/NLRP3 Signaling pathway and activating the Nrf2/HO-1 Signaling pathway.

Varicellovirus bovinealpha 1 (BoAHV-1) is a significant pathogen responsible for respiratory disease in cattle, capable of inducing lung damage independently or co-infection with bacteria. The widespread spread of BoAHV-1 in cattle herds has caused substantial economic losses to the cattle industry. The pathogenic mechanisms of BoAHV-1 are often relevant to robust inflammatory responses, increased oxidative burden, and the initiation of apoptosis. Glycyrrhizin (GLY) is a small-molecule triterpenoid saponin compound obtained from the herb liquorice, which has a broad spectrum of pharmacological properties such as antiviral, anti-inflammatory, and antioxidant effects. Furthermore, GLY regulates lung physiology by modulating oxidative stress, inflammatory response, and cell apoptosis through interference with the NF-κB/NLRP3 and Nrf2/HO-1 Signaling pathways. However, the potential of GLY to mitigate lung injury induced by BoAHV-1 and its underlying mechanism remains unclear. Therefore, in this study, we investigated the protective effect of GLY against pulmonary injury induced by BoAHV-1 in a guinea pig model by reducing viral load and suppressing the inflammatory response, oxidative stress, and apoptosis. The results of this study demonstrated that GLY exerted a protective effect against BoAHV-1-induced lung injury in guinea pigs. Specifically, GLY reduced the levels of pro-inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interleukin (IL)-8 in guinea pig tissues while suppressing the expression of Caspase-1. Additionally, GLY reduced BoAHV-1 load and the number of TUNEL-positive lung cells in guinea pig lungs while inhibiting Caspase 3 protein expression. Furthermore, GLY significantly enhanced lung antioxidant capacity by increasing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity while simultaneously reducing malondialdehyde (MDA) levels. Lung histological observation and score further validated the protective effect of GLY on BoAHV-1-induced lung injury. Furthermore, we observed that the expression of phosphorylated NF-κB p65 (p-NF-κB p65) and NLRP3 proteins in the lung tissue of BoAHV-1-infected guinea pigs decreased after GLY treatment while the expression of Nrf2 and HO-1 proteins increased. These results indicated that GLY inhibited the NF-κB/NLRP3 Signaling pathway and activated the Nrf2/HO-1 Signaling pathway during BoAHV-1 infection. Ultimately, our findings demonstrated that GLY alleviates BoAHV-1-induced inflammation response, oxidative stress, and cell apoptosis by inhibiting the NF-κB/NLRP3 Signaling pathway and activating the Nrf2/HO-1 Signaling pathway to protect guinea pigs from lung injury caused by BoAHV-1. Ultimately, our findings demonstrated that GLY alleviates BoAHV-1-induced inflammation response, oxidative stress, and cell apoptosis by inhibiting the NF-κB/NLRP3 Signaling pathway and activating the Nrf2/HO-1 Signaling pathway to protect guinea pigs from lung injury caused by BoAHV-1. Importantly, this study provides a compelling argument for the GLY in combating respiratory disease in cattle caused by BoAHV-1.