The predictive value of inflammatory biomarkers for major pathological response in non-small cell lung cancer patients receiving neoadjuvant chemoimmunotherapy and its association with the immune-related tumor microenvironment: a multi-center study.

BACKGROUND: Inflammatory biomarkers in the peripheral blood have been established as predictors for immunotherapeutic efficacy in advanced non-small cell lung cancer (NSCLC). Whether they can also predict major pathological response (MPR) in neoadjuvant setting remains unclear. METHODS: In this multi-center retrospective study, 122 and 92 stage I-IIIB NSCLC patients from six hospitals who received neoadjuvant chemoimmunotherapy followed by surgery were included in the discovery and external validation cohort, respectively. Baseline and on-treatment neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR) and systemic immune-inflammation index (SII) were calculated and associated with MPR. Furthermore, resected tumor samples from 37 patients were collected for RNA-sequencing to investigate the immune-related tumor microenvironment. RESULTS: In both the discovery and validation cohorts, the on-treatment NLR, dNLR, PLR, and SII levels were significantly lower in the patients with MPR versus non-MPR. On-treatment SII remained an independent predictor of MPR in multivariate logistic regression analysis. The area under the curve (AUC) of on-treatment SII for predicting MPR was 0.75 (95%CI, 0.67-0.84) in the discovery cohort. Moreover, the predictive value was further improved by combining the on-treatment SII and radiological tumor regression data, demonstrating an AUC of 0.82 (95%CI, 0.74-0.90). The predictive accuracy was validated in the external cohort. Compared with the SII-high group, patients with SII-Low were associated with the activated B cell receptor signaling pathway and a higher intratumoral immune cell infiltration level. CONCLUSIONS: On-treatment SII was independently associated with MPR in NSCLC patients receiving neoadjuvant chemoimmunotherapy. Further prospective studies are warranted.

Establishment of a risk prediction model for prolonged mechanical ventilation after lung transplantation: a retrospective cohort study.

BACKGROUND: Prolonged mechanical ventilation (PMV), mostly defined as mechanical ventilation > 72 h after lung transplantation with or without tracheostomy, is associated with increased mortality. Nevertheless, the predictive factors of PMV after lung transplant remain unclear. The present study aimed to develop a novel scoring system to identify PMV after lung transplantation. METHODS: A total of 141 patients who underwent lung transplantation were investigated in this study. The patients were divided into PMV and non-prolonged ventilation (NPMV) groups. Univariate and multivariate logistic regression analyses were performed to assess factors associated with PMV. A risk nomogram was then established based on the multivariate analysis, and model performance was further examined regarding its calibration, discrimination, and clinical usefulness. RESULTS: Eight factors were finally identified to be significantly associated with PMV by the multivariate analysis and therefore were included as risk factors in the nomogram as follows: the body mass index (BMI, P = 0.036); primary diagnosis as idiopathic pulmonary fibrosis (IPF, P = 0.038); pulmonary hypertension (PAH, P = 0.034); primary graft dysfunction grading (PGD, P = 0.011) at T0; cold ischemia time (CIT P = 0.012); and three ventilation parameters (peak inspiratory pressure [PIP, P < 0.001], dynamic compliance [Cdyn, P = 0.001], and P/F ratio [P = 0.015]) at T0. The nomogram exhibited superior discrimination ability with an area under the curve of 0.895. Furthermore, both calibration curve and decision-curve analysis indicated satisfactory performance. CONCLUSION: A novel nomogram to predict individual risk of receiving PMV for patients after lung transplantation was established, which may guide preventative measures for tackling this adverse event.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BACKGROUND: Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS: Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS: We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS: We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Inhibition of ALOX12-12-HETE Alleviates Lung Ischemia-Reperfusion Injury by Reducing Endothelial Ferroptosis-Mediated Neutrophil Extracellular Trap Formation.

Lung ischemia-reperfusion injury (IRI) stands as the primary culprit behind primary graft dysfunction (PGD) after lung transplantation, yet viable therapeutic options are lacking. In the present study, we used a murine hilar clamp (1 h) and reperfusion (3 h) model to study IRI. The left lung tissues were harvested for metabolomics, transcriptomics, and single-cell RNA sequencing. Metabolomics of plasma from human lung transplantation recipients was also performed. Lung histology, pulmonary function, pulmonary edema, and survival analysis were measured in mice. Integrative analysis of metabolomics and transcriptomics revealed a marked up-regulation of arachidonate 12-lipoxygenase (ALOX12) and its metabolite 12-hydroxyeicosatetraenoic acid (12-HETE), which played a pivotal role in promoting ferroptosis and neutrophil extracellular trap (NET) formation during lung IRI. Additionally, single-cell RNA sequencing revealed that ferroptosis predominantly occurred in pulmonary endothelial cells. Importantly, Alox12-knockout (KO) mice exhibited a notable decrease in ferroptosis, NET formation, and tissue injury. To investigate the interplay between endothelial ferroptosis and NET formation, a hypoxia/reoxygenation (HR) cell model using 2 human endothelial cell lines was established. By incubating conditioned medium from HR cell model with neutrophils, we found that the liberation of high mobility group box 1 (HMGB1) from endothelial cells undergoing ferroptosis facilitated the formation of NETs by activating the TLR4/MYD88 pathway. Last, the administration of ML355, a targeted inhibitor of Alox12, mitigated lung IRI in both murine hilar clamp/reperfusion and rat left lung transplant models. Collectively, our study indicates ALOX12 as a promising therapeutic strategy for lung IRI.

Intestinal microbiota links to allograft stability after lung transplantation: a prospective cohort study.

Whether the alternated microbiota in the gut contribute to the risk of allograft rejection (AR) and pulmonary infection (PI) in the setting of lung transplant recipients (LTRs) remains unexplored. A prospective multicenter cohort of LTRs was identified in the four lung transplant centers. Paired fecal and serum specimens were collected and divided into AR, PI, and event-free (EF) groups according to the diagnosis at sampling. Fecal samples were determined by metagenomic sequencing. And metabolites and cytokines were detected in the paired serum to analyze the potential effect of the altered microbiota community. In total, we analyzed 146 paired samples (AR = 25, PI = 43, and EF = 78). Notably, we found that the gut microbiome of AR followed a major depletion pattern with decreased 487 species and compositional diversity. Further multi-omics analysis showed depleted serum metabolites and increased inflammatory cytokines in AR and PI. Bacteroides uniformis, which declined in AR (2.4% vs 0.6%) and was negatively associated with serum IL-1ß and IL-12, was identified as a driven specie in the network of gut microbiome of EF. Functionally, the EF specimens were abundant in probiotics related to mannose and cationic antimicrobial peptide metabolism. Furthermore, a support-vector machine classifier based on microbiome, metabolome, and clinical parameters highly predicted AR (AUPRC = 0.801) and PI (AUPRC = 0.855), whereby the microbiome dataset showed a particularly high diagnostic power. In conclusion, a disruptive gut microbiota showed a significant association with allograft rejection and infection and with systemic cytokines and metabolites in LTRs.

Lung and gut microbiomes in pulmonary aspergillosis: Exploring adjunctive therapies to combat the disease.

Species within the Aspergillus spp. cause a wide range of infections in humans, including invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis, and are associated with high mortality rates. The incidence of pulmonary aspergillosis (PA) is on the rise, and the emergence of triazole-resistant Aspergillus spp. isolates, especially Aspergillus fumigatus, limits the efficacy of mold-active triazoles. Therefore, host-directed and novel adjunctive therapies are required to more effectively combat PA. In this review, we focus on PA from a microbiome perspective. We provide a general overview of the effects of the lung and gut microbiomes on the growth of Aspergillus spp. and host immunity. We highlight the potential of the microbiome as a therapeutic target for PA.

Improvement of surgical techniques for orthotopic single lung transplantation in rats.

Background: Although orthotopic single lung transplantation in rats has long been established, this model is still highly challenging. Therefore, we made several modifications in anesthesia, lung extraction, vascular clamp, and transplantation procedures for this model. Methods: Fifty cases of rat left lung transplantation were performed using traditional procedures and modified surgical techniques, respectively. Two hundred Sprague Dawley male rats, half as donors and half as recipients, were randomized equally to the two groups. The modifications included orotracheal intubation via a video laryngoscope, retrograde perfusion following anterograde perfusion, a Rummel tourniquet for the occlusion of pulmonary vessels, flushing the vessels and cuffs before anastomosis with heparin, and a simple pleural drainage. The surgical time, warm and cold ischemia time, vascular complications, and survival rate on postoperative day seven were compared between the two groups. Results: The modified surgical techniques significantly reduced the surgical duration (35.7 vs. 46.3 min, P<0.01), warm ischemia time (16.3 vs. 28.8 min, P<0.01), and vascular complications (2% vs. 16%, P=0.04). Moreover, the survival rate on postoperative day 7 was higher in the improved surgical techniques group (96% vs. 80%, P=0.03). Conclusions: We described the improvement of surgical techniques for orthotopic single lung transplantation in rats, which could shorten anastomoses time, reduce vascular complications, and improve survival rate.