Lavage Fluid Research Articles

Abstract A049: Peritoneal tumor DNA as a prognostic biomarker in locally advanced gastroesophageal cancer: A pilot prospective cohort study

Abstract Background: Gastroesophageal cancers commonly spread to the peritoneum. Peritoneal disease significantly alters patient prognosis and treatment-intent. Currently, peritoneal lavage cytology (PLC) is the method of choice for detecting peritoneal micro-metastasis within the peritoneum. Despite its pervasive use, PLC has a variable sensitivity of 10% - 80% in detecting peritoneal micro-metastasis in gastroesophageal cancers. Peritoneal tumor DNA (ptDNA) is tumor-derived DNA detectable in peritoneal lavage fluid. ptDNA-positivity may indicate peritoneal micro- metastasis and may be more sensitive than PLC in staging the peritoneum and predicting disease- free survival (DFS) and overall survival (OS). We aimed to develop and test a tumor-informed platform for ptDNA detection and validate its sensitivity and specificity against PLC. Methods: For this pilot study, peritoneal lavage fluid were collected at staging laparoscopy from 15 patients with gastroesophageal cancer. Cytology and peritoneal metastases (where clinically detectable) were confirmed by histopathology. A tumor-informed ptDNA testing was performed via whole-genome sequencing of tumor tissue and buffy coat to identify somatic variants for each patients, followed by tracking of up to 96 variants in each patient’s peritoneal fluid. Results: 5 out of 15 patients were either cytology positive or had macroscopic peritoneal disease and ptDNA was detectable in all 5 cases. Additionally, out of the 10 cytology-negative patients without macroscopic peritoneal disease, ptDNA positivity was detected in 6 patients prior to commencing neoadjuvant chemotherapy or surgery. Two of these 6 patients demonstrated radiological evidence of recurrence at 15- and 18-months follow-up respectively. Interestingly, one patient with macroscopic peritoneal disease tested negative for cytology. In this case, peritoneal lavage was carried out to the upper two abdominal quadrants only, and the pelvic deposit of disease was detected after the lavage had been completed. Despite a negative cytology result, ptDNA was detected using the same fluid sample, suggesting a higher sensitivity compared to PLC. Conclusions: This pilot study demonstrates that a tumor-informed ptDNA detection platform is feasible and potentially more sensitive than PLC in gastroesophageal cancer. To further validate the clinical utility of ptDNA we are proceeding to a multi-center prospective cohort study. Citation Format: Zexi Allan, Yuxuan Wang, Jeanne Tie, Niall Tebbutt, Nicholas Clemons, Nickolas Papadopoulos, Kenneth Kinzler, Bert Vogelstein, David SH Liu. Peritoneal tumor DNA as a prognostic biomarker in locally advanced gastroesophageal cancer: A pilot prospective cohort study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A049.

Bayesian accuracy estimates for diagnostic tests to detect tuberculosis in captive sun bears (Helarctos malayanus) and Asiatic black bears (Ursus thibetanus) in Cambodia and Vietnam

Effective control of tuberculosis (TB) depends on early diagnosis of disease, yet available tests are unable to perfectly detect infected individuals. In novel hosts diagnostic testing methods for TB are extrapolated from other species, with unknown accuracy. The primary challenge to evaluating the accuracy of TB tests is the lack of a perfect reference test. Here we use a Bayesian latent class analysis approach to evaluate five tests available for ante-mortem detection of pulmonary TB in captive sun bears and Asiatic black bears in Southeast Asia. Using retrospective results from screening of 344 bears at three rescue centres, we estimate accuracy parameters for thoracic radiography, a serological assay (DPP VetTB), and three microbiological tests (microscopy, PCR (Xpert MTB/RIF, Xpert MTB/RIF Ultra), mycobacterial culture) performed on bronchoalveolar lavage samples. While confirming the high specificities (≥ 0.99) of the three microbiological tests, our model demonstrated their sub-optimal sensitivities (<0.7). Thoracic radiography was the only diagnostic method with sensitivity (0.95, 95% BCI: 0.76, 0.998) and specificity (0.95, 95% BCI: 0.91, 0.98) estimated above 0.9. We recommend caution when interpreting DPP VetTB results, with the increased sensitivity resulting from treatment of weakly visible reactions as positive accompanied by a drop in specificity, and we illustrate how the diagnostic value of weak DPP VetTB reactions is particularly reduced if disease prevalence and/or clinical suspicion is low. Conversely, the reduced utility of negative microbiological tests on bronchoalveolar lavage fluid samples when prevalence and/or clinical suspicion is high is demonstrated. Taken together our results suggest multiple tests should be applied and accompanied by consideration of the testing context, to minimise the consequences of misclassification of disease status of bears at risk of TB in sanctuary settings.

Acute eosinophilic pneumonia presented with hemoptysis as the first manifestation

We reported a case of 38-year-old man who was admitted with a 10-day history of hemoptysis. He had reported a significant increasing in tobacco consumption in the week prior to admission. Chest computed tomographic (CT) scans showed multiple ground-glass opacities in bilateral lung fields. The percentage of eosinophils in the bronchoalveolar lavage fluid was over 25%. Based on the patient's imaging findings and the predominant eosinophilia in the bronchoalveolar lavage, a diagnosis of acute eosinophilic pneumonia was made. Systemic corticosteroid therapy was initiated. Repeated CT scan showed the opacities were completely resolved afterwards. Compared with the previous case reports published in Pubmed, which included 234 patients with AEP, the present case described hemoptysis as the only manifestation in an AEP patient, indicating the diversity of clinical manifestations of AEP. Clinicians should consider the probability of AEP in patients with hemoptysis and pulmonary ground-glass opacities after acute tobacco exposure if the disease cannot be explained by other causes.

The Coexistence of Klebsiella pneumoniae and Candida albicans Enhanced Biofilm Thickness but Induced Less Severe Neutrophil Responses and Less Inflammation in Pneumonia Mice Than K. pneumoniae Alone

Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi.

Comparison Between Tracheal Wash and Bronchoalveolar Lavage Cytology for the Assessment of Exercise-Induced Pulmonary Hemorrhage (EIPH) in Racehorses

Exercise-Induced Pulmonary Hemorrhage (EIPH) is a common pulmonary disease among racehorses, diagnosed by the detection of blood in the trachea after strenuous exercise or the presence of hemosiderophages in the bronchoalveolar lavage fluid (BALF). Although the latter is considered the most sensitive method to diagnose EIPH, it is perceived as a less practical and more invasive procedure compared to tracheal wash (TW) collection among racehorse trainers. The present retrospective study aimed to verify the agreement between Tracheal wash and BALF cytology in assessing EIPH in racehorses. For this purpose, cytological data from 172 patients regarding hemosiderophage percentage, hemosiderin score, and percentage of recent, intermediate, and old EIPH were reviewed, and the simplified Total Hemosiderin Score (sTHS) was calculated. Non-parametric statistical tests were used to assess the difference and the correlation between TW and BALF. The two cytological methods strongly agreed in evaluating EIPH in racehorses for hemosiderophage percentage (ρ = 0.89, p < 0.001), hemosiderin score (k = 0.63, p < 0.001), sTHS (ρ = 0.87, p < 0.001), percentage of recent EIPH (ρ = 0.95, p < 0.001), intermediate EIPH (ρ = 0.92, p < 0.001), and old EIPH (ρ = 0.85, p < 0.001). In conclusion, TW showed to be a reliable method, which might substitute BALF in assessing EIPH in racehorses.

Rapid diagnosis of Mycoplasma pneumoniae and prediction of antibiotic resistance by nanopore adaptive sampling

AbstractBackgroundMicrobial infections, particularly in children, require rapid and accurate diagnostics. It is difficult to differentiate pathogens from commensal organisms, and it is impossible to identify antibiotic resistance genes that belong to pathogens with current methods. Third‐generation sequencing provides rapid library preparation and real‐time data acquisition. Nanopore normal sampling (NNS) enables unbiased sequencing of clinical samples without amplification, aiding pathogen identification and antimicrobial resistance gene prediction. However, clinical samples often contain a considerable amount of human DNA, potentially masking pathogen data. Nanopore adaptive sampling (NAS) aims to selectively enrich pathogens, promising improved diagnostics for acute infections and better treatment decisions in clinical practice. This study aimed to determine the utility of NAS in enhancing the real‐time detection of pathogens and predicting AMR in infectious disease outbreaks.MethodsThis study used NAS technology to rapidly and directly detect Mycoplasma pneumoniae infection in bronchoalveolar lavage fluid samples from 28 pediatric patients at Shenzhen Children's Hospital. We assessed the efficacy of NAS compared with that of NNS by evaluating the number of microbial reads and the amount of microbial DNA data. We then compared the accuracy of detecting pathogens between NNS and NAS and between NAS and real‐time polymerase chain reaction assays. Furthermore, we predicted antimicrobial resistance (AMR) and examined AMR genes associated with pathogens.ResultsNAS showed up to a 14.67‐fold increase in the amount of microbial DNA data from patients' samples compared with NNS within the initial 2.5 h of sequencing. Additionally, NAS reduced the amount of host DNA data by up to 6.67‐fold compared with NNS. Unlike TaqMan real‐time polymerase chain reaction assays, NAS technology identified dominant pathogens and provided detailed insight into the abundance of the microbial community. Furthermore, NAS was able to predict AMR profiles of microbial communities and attribute specific AMR traits to individual microbes within the samples.ConclusionThis study shows that NAS advances the clinical diagnosis because it can rapidly detect pathogens directly from patients' samples and provides antimicrobial resistance information for clinical guidance. These abilities further facilitate the application of NAS in personalized treatment, reduce the misuse of broad‐spectrum antibiotics, and promote patients' recovery.

Immunogenicity of intraperitoneal and intranasal liposome adjuvanted VLP vaccines against SARS-CoV-2 infection

Humans get SARS-CoV-2 infection mainly through inhalation; thus, vaccine that induces protective immunity at the virus entry site is important for early control of the infection. In this study, two anionic liposome (L)-adjuvanted VLP vaccines against SARS-CoV-2 were formulated. Baculovirus-Sf21 insect cell system was used for production of VLPs made of full-length S, M and E proteins. S protein of one vaccine (L-SME-VLPs) contained furin cleavage site at the S1/S2 junction, while that of another vaccine (L-S′ME-VLPs) did not. Both vaccines were innocuous and immunogenic when administered IP and IN to mice. Mice immunized IP with L-SME-VLPs/L-S′ME-VLPs (three doses, two-weeks intervals) had serum virus neutralizing (VN) antibodies (in falling order of isotype frequency): IgG3, IgA and IgG2a/IgG3, IgA and IgM, respectively. The L-S′ME VLPs vaccine induced significantly higher serum VN antibody titers than the L-SME-VLPs vaccine. All mice immunized IN with both vaccines had significant rise of VN antibodies in their bronchoalveolar lavage fluids (BALF). The VN antibodies in 67% of immunized mice were Th1- isotypes (IgG2a and/or IgG2b); the immunized mice had also other antibody isotypes in BALF. The intranasal L-S′ME-VLPs should be tested further step-by-step towards the clinical use as effective and safe vaccine against SARS-CoV-2.

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

Early mNGS testing for diagnose and prognostic prediction of early onset pneumonia among in-hospital cardiac arrest patients undergoing extracorporeal cardiopulmonary resuscitation

ObjectivesMetagenomic next-generation sequencing (mNGS) is emerging as a novel diagnostic technology for various infectious diseases; however, limited studies have investigated its application in etiological diagnosis of early onset pneumonia (EOP) among patients undergoing extracorporeal cardiopulmonary resuscitation (ECPR) following in-hospital cardiac arrest (IHCA), The clinical significance of early mNGS in predicting short-term prognosis of IHCA patients after ECPR remains unclear.MethodsThis retrospective study included 76 patients with IHCA who underwent ECPR at the First Affiliated Hospital of Zhengzhou University from January 2018 to December 2022. Baseline characteristics and etiological data of all patients during their hospitalization were collected and statistically analyzed. The primary outcome of this study was the diagnosis of EOP, while the secondary outcomes included successful extracorporeal membrane oxygenation (ECMO) weaning and survival at discharge. Additionally, the characteristics of bronchoalveolar lavage fluid (BALF) flora in these patients were compared by analyzing both mNGS results and culture results.ResultsMultivariate logistic regression were employed to analyze the predictors of ECMO weaning failure, mortality at discharge, and the incidence of EOP. Ultimately, patients with lower SOFA scores on admission [OR (95%CI): 1.447 (1.107-1.890), p=0.007] and those who underwent early mNGS testing within 48 hours after ECPR [OR (95%CI): 0.273 (0.086-0.865), p=0.027] demonstrated a higher probability of successful weaning from ECMO. Patients with higher SOFA scores on admission [OR (95%CI): 2.404 (1.422-4.064), p=0.001], and elevated lactate levels [OR (95%CI): 1.176 (1.017-1.361), p=0.029] exhibited an increased likelihood of mortality at discharge. Furthermore, early mNGS detection [OR (95%CI): 0.186 (0.035-0.979), p=0.047], and lower CRP levels (48h-7d after ECMO) [OR (95%CI):1.011 (1.003-1.019), p=0.006] were associated with a reduced incidence of EOP. In addition, the pathogens detected by mNGS within 48 hours after ECPR were mainly oral colonizing bacteria and viruses, and viruses were in the majority, while all BALF cultures were negative. In contrast, between 48 hours and 7 days after ECPR, BALF cultures were positive in all EOP patients.ConclusionsEarly mNGS testing to identify microbial flora facilitates timely adjustment of antibiotic regimens, thereby reducing the incidence of EOP and improving short-term prognosis in patients undergoing ECPR following IHCA.

V–V ECMO for severe Chlamydia psittaci pneumonia presenting with sudden cardiac arrest: A case report and literature review

Rationale: Psittacosis, also known as parrot fever, is an infectious disease caused by Chlamydia psittaci, which can lead to C psittaci pneumonia. Clinical manifestations are highly nonspecific, which can vary from asymptomatic infection to severe pneumonia and even death. Patient concerns: In this case presentation, we reported one 65-year-old male case of C psittaci pneumonia who was admitted to our hospital on December 2, 2022 due to the chief complaints of poor appetite and fatigue for 3 days as the clinical manifestations. He denied contact with birds but admitted riding horses 1 week ago. Diagnoses: Chlamydia psittaci pneumonia of patient was confirmed through metagenomic sequencing of bronchoalveolar lavage fluid under bronchoscopy. Intervention: Patient was treated with V-V ECMO, invasive mechanical ventilation and CRRT. Outcomes: On December 12, the patient was successfully weaned off V–V ECMO and discharged on December 20, 2022. During postoperative follow-up, CT scan in a local hospital revealed the infiltrative lesions of the lung were absent. Lessons: This case prompts that metagenomic next-generation sequencing is a feasible diagnostic tool for psittacosis, which can rapidly worsen and even cause sudden cardiac arrest. V–V ECMO might be a viable emergency therapeutic option.

Case report: Metagenomic next-generation sequencing for the diagnosis of rare Nocardia aobensis infection in a patient with immune thrombocytopenia

BackgroundNocardiosis poses a diagnostic challenge due to its rarity in clinical practice, non-specific clinical symptoms and imaging features, and the limitations of traditional detection methods. Nocardia aobensis (N. aobensis) is rarely detected in clinical samples. Metagenomic next-generation sequencing (mNGS) offers significant advantages over traditional methods for rapid and accurate diagnosis of infectious diseases, especially for rare pathogens.Case presentationA 52-year-old woman with a history of immune thrombocytopenia for over 2 years was hospitalized for recurrent fever and cough lasting for 10 days. Her initial diagnosis on admission was community-acquired pneumonia, based on chest computed tomography findings of lung inflammation lesion. Empirical treatment with moxifloxacin and trimethoprim-sulfamethoxazole (TMP-SMZ) was initiated. However, her condition failed to improve significantly even after 1 week of treatment. Bronchoalveolar lavage fluid (BALF) subjected to mNGS revealed the presence of N. aobensis, resulting in a diagnosis of pulmonary nocardiosis caused by N. aobensis. This diagnosis was also supported by Sanger sequencing of the BALF. After adjusting the antibiotic regimen to include TMP-SMZ in combination with imipenem, the patient’s condition significantly improved. She was finally discharged with instructions to continue oral treatment with TMP-SMZ and linezolid for 6 months. The patient’s first follow-up 1 month after discharge showed good treatment outcomes but with obvious side effects of the drugs. Consequently, the antibiotic regimen was changed to doxycycline, and the patient continued to improve.ConclusionWe report the first detailed case of pulmonary nocardiosis caused by N. aobensis diagnosed by mNGS. mNGS could be an effective method that facilitates early diagnosis and timely decision-making for the treatment of nocardiosis, especially in cases that involve rare pathogens.

Suppressing neutrophil itaconate production attenuates Mycoplasma pneumoniae pneumonia.

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in which neutrophils play a critical role. Immune-responsive gene 1 (IRG1), responsible for itaconate production, has emerged as an important regulator of inflammation and infection, but its role during M. pneumoniae infection remains unknown. Here, we reveal that itaconate is an endogenous pro-inflammatory metabolite during M. pneumoniae infection. Irg1 knockout (KO) mice had lower levels of bacterial burden, lactate dehydrogenase (LDH), and pro-inflammatory cytokines compared with wild-type (WT) controls after M. pneumoniae infection. Neutrophils were the major cells producing itaconate during M. pneumoniae infection in mice. Neutrophil counts were positively correlated with itaconate concentrations in bronchoalveolar lavage fluid (BALF) of patients with severe M. pneumoniae pneumonia. Adoptive transfer of Irg1 KO neutrophils, or administration of β-glucan (an inhibitor of Irg1 expression), significantly attenuated M. pneumoniae pneumonia in mice. Mechanistically, itaconate impaired neutrophil bacterial killing and suppressed neutrophil apoptosis via inhibiting mitochondrial ROS. Moreover, M. pneumoniae induced Irg1 expression by activating NF-κB and STAT1 pathways involving TLR2. Our data thus identify Irg1/itaconate pathway as a potential therapeutic target for the treatment of M. pneumoniae pneumonia.

Streptococcus salivarius pneumonia-associated pneumomediastinum: a case report and literature review

BackgroundStreptococcus salivarius is an opportunistic pathogen, and there have been no reported cases of Streptococcus salivarius pneumonia to date. Pneumomediastinum is usually secondary to tracheal or esophageal injury and is very rare as a complication of pneumonia. We report a case of Streptococcus salivarius pneumonia complicated by pneumomediastinum, aiming to enhance clinicians’ awareness of rare pathogens and uncommon complications in pneumonia.Case presentationThe patient, a 36-year-old male, presented with a persistent cough and sputum production for one week, accompanied by a sore throat that had developed just one day prior. Chest computed tomography (CT) disclosed pneumomediastinum alongside obstructive atelectasis in the left lower lobe. Streptococcus salivarius infection was conclusively identified through bronchoalveolar lavage metagenomic next-generation sequencing (mNGS), as well as smear and culture analyses. The patient was administered intravenous amoxicillin-clavulanate potassium for a duration of seven days as part of the anti-infection regimen. Given the stability of the patient’s respiratory and circulatory systems, a tube drainage procedure was deemed unnecessary. Post-treatment, the patient’s clinical symptoms notably improved. A subsequent chest CT scan revealed the re-expansion of the left lower lung and near-complete resolution of pneumomediastinum.ConclusionThere are numerous pathogens that can cause pneumonia. While focusing on common pathogens, it is important not to overlook rare ones. When considering infections from rare pathogens, it is recommended to promptly perform a bronchoscopy and submit bronchoalveolar lavage fluid for mNGS to improve pathogen detection rates. During the diagnosis and treatment of pneumonia, it is crucial to be vigilant for rare complications. When a patient presents with symptoms such as dyspnea or subcutaneous emphysema, it is advisable to immediately perform a chest CT scan to rule out pneumomediastinum.

High utility of bronchoalveolar lavage fluid metagenomic next-generation sequencing approach for etiological diagnosis of pneumonia

BackgroundFor patients with pneumonia, the rapid detection of pathogens is still a major global problem in clinical practice because traditional diagnostic techniques for infection are time-consuming and insensitive. Metagenomic next-generation sequencing (mNGS) is a novel technique that has the potential to improve pathogen diagnosis. This study aimed to investigate the microbiological diagnostic ability of mNGS compared with conventional culture and to determine the optimal time to test patients for pneumonia.MethodsA prospective study using data from June 2020 to June 2021 was performed at a tertiary teaching hospital in China. We included 56 patients from all adult patients with a clinical diagnosis of pneumonia. Blood and bronchoalveolar lavage fluid (BALF) samples were taken for simultaneous mNGS and conventional culture testing.ResultsAll 56 patients underwent both conventional culture and mNGS. Of these patients, 37 were diagnosed with severe pneumonia and 17 were diagnosed with non-severe pneumonia. The top three pathogenic bacteria detected by mNGS were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Enterococcus faecium was detected more frequently in the non-severe pneumonia group (4 vs. 0, p < 0.05). The findings revealed that the detection rate of mNGS (84%) was superior to that of conventional culture methods (48%). Notably, the percentage of mNGS-positive BALF samples (46/56, 82.14%) was significantly greater than that of blood samples (27/56, 48.21%). The etiological comparison demonstrated that mNGS-positive samples, which received clinical approval, tended to be associated with a more normalized temperature, lower PCO2 levels, and a higher SOFA score than mNGS-negative samples (p = 0.022, p = 0.0.028, and p = 0.038, respectively).ConclusionsIn this study, we discovered that the etiology of lung infections frequently involves multiple pathogens. The use of mNGS in BALF is instrumental for detecting nonviral pathogens associated with lung infections. Although the rate of positive blood NGS results is significantly influenced by various clinical factors, for patients suspected of having viral, Legionella, or tsutsugamushi infections, plasma mNGS could serve as a complementary diagnostic tool.

Oxidative stress in the alveolar lavage fluid of children with Mycoplasma pneumoniae pneumonia.

To investigate the correlation between oxidative stress in the bronchoalveolar lavage fluid (BALF) of children with Mycoplasma pneumoniae pneumonia (MPP) and the clinical characteristics of severe MPP (SMPP) and refractory MPP (RMPP). Clinical and BALF-related data were collected from 83 patients with MPP, of which 29 had SMPP and 54 had general MPP (GMPP); 37 patients were in the RMPP group and 46 in the non-RMPP group. The levels of malondialdehyde (MDA) and advanced oxidation protein products (AOPP) as well as the activity levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in BALF were detected. Logistic regression analyses were performed on MDA, AOPP, SOD, GSH-PX, gender, heat peak, neutrophil percentage, C-reactive protein, lactate dehydrogenase, d-dimer, lung consolidation, sputum embolus, and pleural effusion. The levels of MDA and AOPP in the BALF of the MPP group were significantly higher than those in the control group (p < .05), whereas SOD and GSH-PX levels were lower than those in the control group (p < .05). The BALF AOPP levels in the RMPP group were higher than those in the non-RMPP group, and the SOD and GSH-PX levels in the BALF were lower than those in the non-RMPP group; the difference was statistically significant (p < .05). The levels of MDA and AOPP in the BALF of children in the SMPP group were higher than those in the GMPP group, and the levels of SOD and GSH-PX were lower than those in the GMPP group, with statistically significant differences (p < .05). The C-index of the logistic regression model was 0.960 (95% confidence interval 0.958-0.963), which indicates that the model has good predictive ability. Advanced oxidation protein products may be a marker for predicting the conditions of SMPP and RMPP, and the prediction model can assess the risk of progression in children to RMPP, which is conducive to clinical diagnosis and treatment.

Dietary Eicosapentaenoic Acid Improves Ozone-Induced Pulmonary Inflammation in C57/BL6 Mice

Ambient concentrations of the air pollutant, ozone, are rising with increasing global temperatures. Ozone is known to increase incidence and exacerbation of chronic lung diseases, which will increase as ambient ozone levels rise. Studies have identified diet as a variable able to modulate the pulmonary health effects associated with ozone exposure. Eicosapentaenoic Acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) consumed through diet that lowers inflammation through conversion to oxylipins including Hydroxy Eicosapentaenoic Acids (HEPEs). However, the role of dietary EPA in ozone-induced pulmonary inflammation is unknown. Therefore, we hypothesize increasing dietary EPA will decrease ozone-induced pulmonary inflammation and injury through the production of HEPEs. To test this, male C57BL/6J mice were fed a purified control diet or EPA-supplemented diet for 4 weeks and then exposed to filtered air (FA) or 1 part per million ozone for 3 hours. 24 or 48 hours post exposure, bronchoalveolar lavage (BAL) fluid was collected to assess airspace inflammation/injury and lung tissue was collected for targeted Liquid Chromatography-Mass Spectrometry (LC-MS/MS) lipidomics. Following ozone exposure, EPA supplementation did not alter markers of lung injury, but decreased ozone-induced airspace neutrophilia. LC-MS/MS targeted lipidomics reveal dietary EPA supplementation increased pulmonary EPA-derived metabolites including 5-HEPE and 12-HEPE. Additionally, EPA supplementation decreased pulmonary levels of pro-inflammatory Arachidonic Acid (AA)-derived metabolites. To evaluate if dietary EPA reduces ozone-induced pulmonary inflammation through increased pulmonary HEPEs, C57BL/6J mice were administered 5- and 12-HEPEs systemically prior to filtered air or ozone exposure. Pretreatment with 5- and 12-HEPEs reduced ozone-driven increases in airspace macrophages. Together, these data indicate that an EPA-supplemented diet protects against ozone-induced airspace inflammation which is, in part, due to increasing pulmonary concentrations of 5- and 12-HEPEs. These findings suggest that dietary EPA and/or increasing EPA-derived metabolites in the lung can reduce ozone-driven incidences and exacerbations of chronic pulmonary diseases.

Aerosolized Harmful Algal Bloom Toxin Microcystin-LR Induces Type 1/Type 17 Inflammation of Murine Airways

Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice; however, little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1- and Th17-driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory and pro-resolving oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research, particularly into populations with predispositions to type 1/type 17 inflammation.

Characterization of a Mycoplasma hyopneumoniae aerosol infection model in pigs

The purpose of the present study was to develop and characterize an experimental aerosol model for Mycoplasma hyopneumoniae (M. hyopneumoniae) infection and respiratory disease in pigs. The experiment was carried out to determine the pathogenicity, colonization, mucosal immune response, and clinical course of disease of dose-controlled aerosols of M. hyopneumoniae. Four groups of three M. hyopneumoniae-free gilts each were individually exposed to aerosols of diluted lung homogenate containing M. hyopneumoniae strain 232 in a chamber. Each group was exposed to different doses of viable organisms (105 to 106 color changing units/mL during 15-20 or 30-35min in two consecutive days). Nasal, laryngeal, and deep-tracheal secretions were collected from each gilt at 0, 7, 14, 21, and 28 days post-exposure (dpe). Blood samples were collected at 0 and 28 dpe. At necropsy, lung lesions were assessed, and bronchial secretions and bronchoalveolar lavage fluid (BALF) were collected from each lung set. Blood was used to assess seroconversion by means of an indirect ELISA, while BALF, deep-tracheal and nasal secretions were tested by modifying the ELISA to evaluate mucosal IgG and IgA production. Nasal, laryngeal, deep-tracheal, and bronchial secretions were tested by real-time PCR to evaluate bacterial load. Gilts became infected irrespective of the infectious dose, as determined by M. hyopneumoniae detection in deep-tracheal secretions from all gilts at 7 dpe. A specific local humoral immune response starting at 14 dpe was detected in all gilts. While all experimental groups presented gilts with some extent of mycoplasmal pneumonia, only the exposure of gilts to high-dose aerosols consistently reproduced typical clinical signs and severe lung lesions. These results showed that the reproduction of mycoplasmal pneumonia by means of infectious aerosols can be successfully achieved at experimental level, making this model a valuable alternative to evaluate preventive and treatment measures against M. hyopneumoniae.

Salidroside attenuates sepsis-induced acute lung injury by inhibiting ferroptosis-dependent pathway.

Sepsis triggers a systemic inflammatory response that can lead to acute lung injury (ALI). Salidroside (SAL) has many pharmacological activities such as antiinflammatory and anti-oxidation. The objective of the study was to explore the mechanism of SAL on ALI caused by sepsis. A model of ALI in septic mice was established by cecal ligation and puncture. Following SAL treatment, the effect of SAL on the ferroptosis pathway in mice was analyzed. The pathological damage of lung tissue, the levels of inflammatory factors and apoptosis in bronchoalveolar lavage fluid (BALF) of mice were evaluated, and the changes of gene expression level and metabolite content abundance were explored by combining transcriptomics and metabolomics analysis. The effect of SAL on ferroptosis in mice with lung injury was observed by intraperitoneal injection of ferroptosis activator Erastin or ferroptosis inhibitor Ferrostatin-1 to promote or inhibit ferroptosis in mice. SAL significantly alleviated the pathological damage of lung tissue, decreased the number of TUNEL positive cells and the levels of TNF-α, IL-1β, IL-6 in BALF, and increased the level of IL- 10 in lung injury mice. Moreover, the Fe2+ content and malondialdehyde decreased significantly, the reactive oxygen species and glutathione content increased significantly, and the arachidonic acid metabolites 20-hydroxyeicosatetraenoic acid (20- HETE), (5Z, 8Z, 10E, 14Z)-12-Oxoeicosa-5,8,10,14-tetraenoic acid (12-OxOETE), (5Z, 8Z, 10E, 14Z)-(12S)-12-Hydroxyeicosa-5,8,10,14-tetraenoic acid (12(S)-HETE), (5Z, 8Z, 14Z)-11,12-Dihydroxyeicosa-5,8,14-trienoic acid (11,12-DHET), (5Z, 11Z, 14Z)-8,9- Dihydroxyeicosa-5,11,14-trienoic acid, Leukotriene B4, Leukotriene D4 were significantly up-regulated after SAL treatment. Salidroside alleviates ALI caused by sepsis by inhibiting ferroptosis.

Effect of vaccination against Mycoplasma hyopneumoniae on divergent pig genetic groups

The bacterium Mycoplasma hyopneumoniae (Mhp) causes a chronic infectious respiratory disease in pigs, leading to important economic losses. This study aimed to compare the immune response of the local Piau breed and a commercial line to Mhp vaccination. For this, two phases were carried out. In the first, gene expression of toll-like receptors (TLR2, TLR4, TLR6, and TLR10) and cytokines (IL2, IL6, IL8, IL10, IL12, IL13, TNFα, and TGFβ) was assessed in porcine blood mononuclear cells (PBMC) from the two genetic groups before and after vaccination. In the second experiment, nitric oxide production, specific antibodies, and gene expression of toll-like receptors and cytokines were evaluated in bronchoalveolar lavage fluid (BALF) cells of vaccinated and unvaccinated pigs. After vaccination against Mhp, TLR2, TLR4, TLR6, TLR10, IL6, TNFα, and TGFβ expression levels were elevated in PBMC from commercial animals, and TLR6, TLR10, and TGFβ expression levels were elevated in PBMC from the Piau group. Vaccination also increased the production of Mhp-specific IgG antibodies in BALF cells in the Piau breed. Comparison of the two genetic groups revealed differences in TNFα and IL10 expression in BALF cells. These results show that Piau pigs have different immune responses to vaccination compared with commercial animals. It is worth noting that these genetic differences between both genetic groups may be related to phenotypic differences in Mhp resistance or susceptibility.