Clinical findings and outcome predictors for multinodular pulmonary fibrosis in horses: 46 cases (2009-2019).

BACKGROUND: Prognostic indicators for equine multinodular pulmonary fibrosis (EMPF), an interstitial fibrosing lung disease, are poorly described. HYPOTHESIS/OBJECTIVES: Describe diagnostic findings and outcome predictors for EMPF. ANIMALS: Forty-six adult horses with EMPF. METHODS: Retrospective multicenter case series from 2009 to 2019. Radiographic (n = 27) and ultrasonographic studies (n = 19) from EMPF horses and bronchoalveolar lavage fluid (BALF) cytology from 6 EMPF and 13 asthma cases were independently reviewed and blinded to diagnosis and outcome. Associations between predictor variables and survival were assessed by predictor screening followed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Primary clinical findings were weight loss (36/46, 78%), increased respiratory effort (33/46, 72%), tachypnea (32/46, 70%), and fever (18/46, 39%). Macrophage atypia was seen in more EMPF than asthmatic horse BALF (67% vs. 8%; P = .02). Equine herpesvirus 5 (EHV-5) was detected in 24 of 30 (80%) and hyperfibrinogenemia in 25 of 28 (89%) cases. Twenty-seven of 46 horses (59%) and 11 of 45 (24%) survived to discharge and to 3 months, respectively. Three-month survival was associated with lower median (range) respiratory rates (30 [24-36] vs. 41 [30-60] breaths per minute; P = .04), and higher BALF lymphocyte:neutrophil ratios (4.7 [1.4-22] vs. 0.47 [0.11-1.9]; P = .01) and blood lymphocyte counts (1.25 [0.93-2.55] vs. 0.90 [0.70-1.24] × 109/L; P = .03). Imaging findings, EHV-5 detection, and corticosteroid treatment were not associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Fever is not a sensitive clinical sign of EMPF. Diagnostic testing should be pursued for horses with increased respiratory rate and effort and weight loss. The prognosis for EMPF horses is poor. Corticosteroid treatment does not improve 3-month survival.

Exploratory mass cytometry analysis reveals immunophenotypes of cancer treatment-related pneumonitis.

Anticancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

Dynamic shifts in lung cytokine patterns in post-COVID-19 interstitial lung disease patients: a pilot study.

Introduction: The pathogenesis of post-COVID interstitial lung disease, marked by lung tissue scarring and functional decline, remains largely unknown. Objectives: We aimed to elucidate the temporal cytokine/chemokine changes in bronchoalveolar lavage (BAL) from patients with post-COVID interstitial lung disease to uncover potential immune drivers of pulmonary complications. Design: We evaluated 16 females diagnosed with post-COVID interstitial lung disease, originating from moderate to severe cases during the second epidemic wave in the Autumn of 2020, treated at the Pneumology Department of the Arad County Clinical Hospital, Romania. Their inflammatory response over time was compared to a control group. Methods: A total of 48 BAL samples were collected over three intervals (1, 3, and 6 months) and underwent cytology, gene, and protein expression analyses for pro/anti-inflammatory lung cytokines and chemokines using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results: One month after infection, there were significant increases in the levels of IL-6 and IL-8. These levels decreased gradually over the course of 6 months but were still higher than those seen in control. Interferon-gamma and tumor necrosis factor alpha exhibited similar patterns. Persistent elevations were found in IL-10, IL-13, and pro-fibrotic M2 macrophages' chemokines (CCL13 and CCL18) for 6 months. Furthermore, pronounced neutrophilia was observed at 1 month post-COVID, highlighting persistent inflammation and lung damage. Neutrophil efferocytosis, aiding inflammation resolution and tissue repair, was evident at the 1-month time interval. A notable time-dependent reduction in CD28 was also noticed. Conclusion: Our research provides insight into the immunological processes that may lead to the fibrotic changes noted in the lungs following COVID-19.

BACKGROUND: Post-COVID lung disease represents a significant health concern that demands comprehensive research. The pathogenesis of post-COVID interstitial lung disease, marked by lung tissue scarring and functional decline, remains largely unknown. METHODS: We evaluated 16 females diagnosed with post-COVID interstitial lung disease, originating from moderate to severe cases during the second epidemic wave in the Autumn 2020, treated at the Pneumology Department of the Arad County Clinical Hospital, Romania. Their inflammatory response over time was compared to a control group. A total of 48 BAL samples were collected over three intervals (1, 3, and 6 months) and underwent cytology, gene, and protein expression analyses for pro/anti-inflammatory lung cytokines and chemokines using RT-PCR and ELISA The interrelationships between the expression levels of various pro-inflammatory and anti-inflammatory cytokines and chemokines by Pearson's correlations was investigated. RESULTS: One month after infection, there were significant increases in the levels of IL-6 and IL-8. These levels decreased gradually over the course of six months but were still higher than those seen in control. IFN-γ and TNF-α exhibited similar patterns. Persistent elevations were found in IL-10, IL-13, and pro-fibrotic M2 macrophages' chemokines (CCL13 and CCL18) for six months. Pronounced neutrophilia was observed at 1 month post-COVID, highlighting persistent inflammation and lung damage. Neutrophils efferocytosis, aiding inflammation resolution and tissue repair, was evident at the 1-month time-interval. A notable time-dependent reduction in CD28 was also noticed. CONCLUSIONS: Our research provides insight into the immunological processes that may lead to the fibrotic changes noted in the lungs following COVID-19.

Dynamic shifts in lung cytokine patterns in post-COVID-19 interstitial lung disease patients: a pilot study The objective of this pilot study was to investigate changes in lung cytokine pro- and anti-inflammatory profiles among patients with interstitial lung disease after COVID-19 infection.

The agreement between bronchoalveolar lavage, bronchial wash and sputum culture: a retrospective study.

PURPOSE: Bronchoalveolar lavage is commonly used in clinical practice for unresolved pneumonia. However, bronchoalveolar lavage is not suitable for all patients as it is an invasive procedure and can worsen oxygenation. The diagnostic value of bronchial wash and sputum has been debated extensively over the years. In this study, we aim to compare the diagnostic value in several pathogens of bronchoalveolar lavage and bronchial wash, and secondarily bronchoalveolar lavage and sputum. METHODS: We retrospectively included all adult patients in our hospital who underwent bronchoalveolar lavage, bronchial wash, and where sputum sampling was done between January 1st of 2018 and December 31st of 2021. The intraclass correlation coefficient was computed for the three tests. RESULTS: In total, 308 patients were included. We found a level of correlation of 0.819 and 0.865, respectively, between bronchoalveolar lavage and bronchial wash for two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. For Stenotrophomonas maltophilia and Aspergillus fumigatus, we found an intraclass correlation coefficient of 0.568 and 0.624, respectively. Between bronchoalveolar lavage and sputum, we found varying levels of agreement. CONCLUSION: Our study shows reasonably well agreement levels between bronchoalveolar lavage and bronchial wash, suggesting that bronchial wash could potentially be an alternative to bronchoalveolar lavage.

Alveolar cytokines and interferon autoantibodies in COVID-19 ARDS.

Background: Type I interferon (IFN-I) and IFN autoantibodies play a crucial role in controlling SARS-CoV-2 infection. The levels of these mediators have only rarely been studied in the alveolar compartment in patients with COVID-19 acute respiratory distress syndrome (CARDS) but have not been compared across different ARDS etiologies, and the potential effect of dexamethasone (DXM) on these mediators is not known. Methods: We assessed the integrity of the alveolo-capillary membrane, interleukins, type I, II, and III IFNs, and IFN autoantibodies by studying the epithelial lining fluid (ELF) volumes, alveolar concentration of protein, and ELF-corrected concentrations of cytokines in two patient subgroups and controls. Results: A total of 16 patients with CARDS (four without and 12 with DXM treatment), eight with non-CARDS, and 15 healthy controls were included. The highest ELF volumes and protein levels were observed in CARDS. Systemic and ELF-corrected alveolar concentrations of interleukin (IL)-6 appeared to be particularly low in patients with CARDS receiving DXM, whereas alveolar levels of IL-8 were high regardless of DXM treatment. Alveolar levels of IFNs were similar between CARDS and non-CARDS patients, and IFNα and IFNω autoantibody levels were higher in patients with CARDS and non-CARDS than in healthy controls. Conclusions: Patients with CARDS exhibited greater alveolo-capillary barrier disruption with compartmentalization of IL-8, regardless of DXM treatment, whereas systemic and alveolar levels of IL-6 were lower in the DXM-treated subgroup. IFN-I autoantibodies were higher in the BALF of CARDS patients, independent of DXM, whereas IFN autoantibodies in plasma were similar to those in controls.

Performance of the Galactomannan Test for the Diagnosis of Invasive Pulmonary Aspergillosis using Non-invasive Proximal Airway Samples.

OBJECTIVE: To diagnose invasive pulmonary aspergillosis (IPA), galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (BALF) is widely used. However, the utility of proximal airway GM test (from induced sputum or tracheal aspirate) has not been well elucidated. METHODS: In this retrospective cohort study, we evaluated the diagnostic performance of proximal airway GM in diagnosis of IPA including COVID-19 associated pulmonary aspergillosis (CAPA). Between January 2022 and January 2023, patients who had been tested for GM with clinical suspicion or for surveillance from any specimen (serum, induced sputum, tracheal aspirate, and BALF) were screened. IPA was diagnosed using EORTC/MSGERC criteria, and CAPA was diagnosed following the 2020 ECMM/ISHAM consensus criteria. RESULTS: Of 624 patients with GM results, 70 met the criteria for proven/probable IPA and 427 had no IPA. The others included possible IPA and chronic form of aspergillosis. The sensitivities and specificities of serum, proximal airway, and BALF GM for proven/probable IPA versus no IPA were 78.9% and 70.6%, 93.1% and 78.7%, and 78.6% and 91.0%, respectively. Areas under the receiver operating characteristic curve (AUCs) were 0.742 for serum GM, 0.935 for proximal airway GM, and 0.849 for BALF GM (serum GM vs proximal airway GM, p = 0.014; proximal airway GM vs BALF GM, p = 0.334; serum GM vs BALF GM, p = 0.286). CONCLUSION: This study demonstrates that the performance of GM test from non-invasive proximal airway samples is comparable or even better than those from serum and distal airway sample (BALF).

Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections.

Circular RNAs (circRNAs) are a class of transcripts that often are generated by back-splicing that covalently connects the 3'end of the exon to the 5'end. CircRNAs are more resistant to nuclease and more stable than their linear counterparts. One of the well-recognized roles of circRNAs is the miRNA sponging effects that potentially lead to the regulation of downstream proteins. Despite that circRNAs have been reported to be involved in a wide range of human diseases, including cancers, cardiovascular, and neurological diseases, they have not been studied in inflammatory lung responses. Here, we analyzed the circRNA profiles detected in extracellular vesicles (EVs) obtained from the broncho-alveolar lavage fluids (BALF) in response to LPS or acid instillation in mice. Next, we validated two specific circRNAs in the BALF-EVs and BALF cells in response to endotoxin by RT-qPCR, using specific primers targeting the circular form of RNAs rather than the linear host RNAs. The expression of these selected circRNAs in the BALF inflammatory cells, alveolar macrophages (AMs), neutrophils, and lung tissue were analyzed. We further predicted the potential miRNAs that interact with these circRNAs. Our study is the first report to show that circRNAs are detectable in BALF EVs obtained from mice. The EV-cargo circRNAs are significantly altered by the noxious stimuli. The circRNAs identified using microarrays may be validated by RT-qPCR using primers specific to the circular but not the linear form. Future studies to investigate circRNA expression and function including miRNA sponging in lung inflammation potentially uncover novel strategies to develop diagnostic/therapeutic targets.

Illumina and Nanopore sequencing in culture-negative samples from suspected lower respiratory tract infection patients.

Objective: To evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests. Methods: Patients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms. Results: Thirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs. 93.5% and 42.9 vs. 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium, and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs. 78.6%, 36.4% vs. 90.9%, and 50% vs. 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs. 77.8% and 42.9% vs. 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/42) based on pathogen diagnosis. Conclusion: Both platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection.

Quantitative evaluation of bronchoalveolar lavage for the treatment of Severe mycoplasma pneumoniae pneumonia in children-A new complementary index: Bronchial Insufflation Sign Score.

OBJECTIVE: The aim of this study was to investigate the value of Broncoplasma Insufflation Sign in lung ultrasound signs in assessing the efficacy of bronchoalveolar lavage in Severe mycoplasma pneumoniae pneumonia in children. METHODS: Forty-seven children with Severe mycoplasma pneumoniae pneumonia were treated with medication and bronchial lavage. Laboratory and imaging results were collected, and lung ultrasonography was performed before bronchoalveolar lavage and 1, 3, and 7 days after lavage to record changes in Bronchial Insufflation Sign and changes in the extent of solid lung lesions. Factors affecting the effectiveness of bronchoalveolar lavage were analyzed using logistic regression and other factors. RESULTS: Bronchial Insufflation Sign Score and the extent of lung solid lesions were the factors affecting the effectiveness of bronchoalveolar lavage treatment. The smaller the area of lung solid lesions and the higher the Bronchial Insufflation Sign Score, the more effective the results of bronchoalveolar lavage treatment were, and the difference was statistically significant, with a difference of p < 0.05. The Bronchial Insufflation Sign Score had the highest sensitivity and specificity for the prediction of the efficacy of bronchoalveolar lavage treatment in the first 7 days after the treatment. CONCLUSION: Bronchial Insufflation Sign Score combined with the extent of solid lung lesions can assess the efficacy of bronchoalveolar lavage in the treatment of Severe mycoplasma pneumoniae pneumonia in children; lung ultrasound is a timely and effective means of assessing the efficacy of bronchoalveolar lavage.

PCR diagnostic platforms for non-Aspergillus mold infections: ready for routine implementation in the clinic?

INTRODUCTION: While Aspergillus spp. remain the predominant cause of invasive mold infections, non-Aspergillus molds, such as the Mucorales or Fusarium spp., account for an increasing proportion of cases. The diagnosis of non-Aspergillus invasive mold infections (NAIMI) is challenging because of the low sensitivity and delay of conventional microbiological tests. Therefore, there is a particular interest to develop molecular tools for their early detection in blood or other clinical samples. AREAS COVERED: This extensive review of the literature discusses the performance of Mucorales-specific PCR and other genus-specific or broad-range fungal PCR that can be used for the diagnosis of NAIMI in diverse clinical samples, with a focus on novel technologies. EXPERT OPINION: PCR currently represents the most promising approach, combining good sensitivity/specificity and ability to detect NAIMI in clinical samples before diagnosis by conventional cultures and histopathology. Several PCR assays have been designed for the detection of Mucorales in particular, but also Fusarium spp. or Scedosporium/Lomentospora spp. Some commercial Mucorales PCRs are now available. While efforts are still needed for standardized protocols and the development of more rapid and simpler techniques, PCR is on the way to becoming an essential test for the early diagnosis of mucormycosis and possibly other NAIMIs.

Case series of complicated bronchopulmonary lophomoniasis. Differential diagnosis of tuberculosis?

Pulmonary lophomoniasis is a rare and life-threatening disease, most commonly reported across Asian and Latin American countries. Here, we have reported two cases of pulmonary lophomoniasis presenting with atypical manifestations. Case #1 represents a 19-year-old male patient with clinical characteristics suggestive of tuberculosis, presenting with hemoptysis and receiving antituberculosis treatment. Case #2 represents a 69-year-old man with post-tuberculosis pulmonary disease with cystic bronchiectasis presenting with polymicrobial co-infection. Based on our case experience, lophomoniasis should be considered in patients with pneumonia who do not respond to antibiotic treatment, and the corresponding epidemiological factors should be carefully considered in addition to bronchoscopy for precise diagnosis.

Identification of the major immune differences in severe asthmatic children according to their atopic dermatitis status.

Severe asthma (SA) affects 2% to 5% of asthmatic children. Atopic dermatitis can affect up to 34% of children with SA (cwSA). Atopic dermatitis and asthma share common genetic and immunological features. However, not all children with SA suffer from AD, and it remains unclear whether the overall immune profiles of these children are similar. In this study, seventeen cwSA (9.8 [7.1-13.2] years; seven with and ten without AD) were enrolled. Bronchoalveolar lavage (BAL) and blood samples were collected from these patients. Seventy-three cytokines/chemokines and distinct immune T cell populations were evaluated in blood and BAL. We found that BAL and blood immune profiles of cwSA with and without AD were globally similar. However, specific differences were observed, namely lower frequency of Tc2, Th17 and IL-17-producing mucosal associated invariant T (MAIT-17) cells and higher CD8/CD4 ratio and IL-22 concentrations in BAL and of CCL19 concentrations in plasma from cwSA with AD. Further, in contrast with cwSA without AD, we found a positive correlation between a set of plasma cytokines and almost all cytokines in BAL in cwSA with AD. In conclusion, this study shows the major immune differences between cwSA with and without AD in BAL and blood suggesting that distinct endotypes may be implicated in the inflammatory responses observed in these pediatric patients.

Spectrum of pathogens and their antimicrobial susceptibility pattern isolated from bronchoalveolar lavage specimens at a hilly area in Uttarakhand.

Background: Lower respiratory tract infections (LRTIs) are associated with significant morbidity and mortality in all age groups, especially young children and the elderly population. Various gram-positive and gram-negative organisms such as Streptococcus spp., Pseudomonas spp., and Klebsiella spp. have been implicated as a pathogen in bronchoalveolar lavage (BAL) specimens collected from such patients. Aims and Rationale of the Study: The present study is aimed at assessing the spectrum of the bacterial isolates and determining the antimicrobial resistance obtained from the BAL fluid from admitted patients at various wards and intensive care units (ICUs) of a tertiary care hospital in Dehradun. This will be the stepping stone in our efforts toward becoming a future antimicrobial steward and framing local antibiograms based on such data. Material and Methods: Two hundred BAL specimens were collected from patients admitted to various wards and ICUs of the hospital who were suffering from LRTI. The BAL specimens were subjected to direct microscopy and culture. Identification and susceptibility testing were performed. Results: The most predominant isolates were Pseudomonas aeruginosa (16/39 (41.02%)) followed by Klebsiella pneumoniae (7/39 (17.94%)) and Acinetobacter spp. (6/39 (15.38%)). Sixty-five percentage of Pseudomonas aeruginosa, 71% of Klebsiella pneumoniae, and 83% of Acinetobacter spp. showed intermediate results with colistin. Conclusion: Nonfermenters constitute a significant group of organisms isolated from bronchoalveolar lavage (BAL) specimens in patients with lower respiratory tract infections (LRTIs). Hence, it is extremely important to correctly identify and determine the resistance pattern of such isolates so that appropriate empirical therapy can be initiated for the benefit of the patient.

Porphyromonas gingivalis-induced hematogenous disseminated severe pneumonia: a case report.

Porphyromonas gingivalis, a gram-negative oral anaerobe among more than more than 500 bacterial species that colonizing the oral cavity, is involved in the pathogenesis and prototypic polybacterial consortium of periodontitis. It is mainly found in oral infections and rarely present in other organ diseases. Here, we describe a 43-year-old man with underlying diabetes who developed hematogenous disseminated severe pneumonia after P. gingivalis had invaded the blood. Next-generation sequencing of early alveolar lavage fluid and blood samples confirmed the diagnosis. The patient's lung infection improved after targeted antimicrobial treatment. He was successfully weaned from ventilatory support and transferred to the general ward. This case illustrates bacterial entry into the bloodstream of a patient with diabetes who had periodontal disease but did not maintain oral hygiene, leading to severe pneumonia. Periodontal disease is often ignored by the public, and it is difficult for critical care physicians to link severe pneumonia with periodontal disease. Thus, this case represents an important warning to critical care clinicians.

Pneumonia Caused by Chlamydia psittaci and SARS-CoV-2 Coinfection Diagnosed Using Metagenomic Next-Generation Sequencing: A Case Report.

We report a case of pneumonia caused by coinfection with Chlamydia psittaci and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB.1 variant, confirmed using metagenomic next-generation sequencing (mNGS) and quantitative polymerase chain reaction (qPCR). C. psittaci and SARS-CoV-2 were detected in bronchoalveolar lavage fluid using mNGS. Additionally, mNGS detected C. psittaci in blood and nasopharyngeal specimens and was more sensitive than qPCR. The patient recovered after treatment with moxifloxacin. This report highlights the use of coinfections of C. psittaci and SARS-CoV-2, as mNGS has already been recognized to be a diagnostic tool for identifying coinfections.

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (µCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this µCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. SIGNIFICANCE: This innovative µCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Murine Models of Cryptococcus Infection.

Cryptococcus is recognized as one of the emerging fungal pathogens that have major impact on diverse populations worldwide. Because of the high mortality rate and limited antifungal therapy options, there is an urgent need to understand the impact of dynamic processes between fungal pathogens and hosts that influence cryptococcal pathogenesis and disease outcomes. With known common limitations in human studies, experimental murine cryptococcosis models that can recapitulate human disease provide a valuable tool for studying fungal virulence and the host interaction, leading to development of better treatment strategies. Infection with Cryptococcus in mice via intranasal inhalation is mostly used because it is noninvasive and considered to be the most common mode of infection, strongly correlating with cryptococcal disease in humans. The protocols described in this article provide the procedures of establishing a murine model of Cryptococcus infection by intranasal inhalation and assessing the host immune response and disease progression during Cryptococcus infection. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Murine model of pulmonary cryptococcal infection via intranasal inhalation Basic Protocol 2: Assessment of the pulmonary immune response during Cryptococcus infection Support Protocol: Evaluation of pulmonary gene expression by real-time PCR Basic Protocol 3: Enumeration of survival rate and organ fungal burden.

Macrophage and CD8 T cell discordance are associated with acute lung allograft dysfunction progression.

BACKGROUND: Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. METHODS: We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. RESULTS: BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p < 0.05 for each). Recipients with discordance between these cells had a nearly 5-fold increased risk of severe graft dysfunction or death (HR 4.6, 95% CI 1.1-19.2, adjusted p = 0.03). We validated these key findings in 2 public lung transplant genomic datasets. CONCLUSIONS: BAL anti-inflammatory macrophages may protect against CLAD by suppressing CD8 T cells. These populations merit functional and longitudinal assessment in additional cohorts.

Bird-related non-fibrotic hypersensitivity pneumonitis with bronchoalveolar lavage fluid eosinophilia that developed after COVID-19 vaccination: A case report.

A 60-year-old man who had been keeping seven budgerigars and four cockatiels in his house for 2 years developed dyspnea and was admitted to our hospital the day after receiving the second dose of the messenger RNA coronavirus disease 2019 vaccination. Chest high resolution computed tomography (HRCT) showed bilateral ground glass opacities without nodules or mosaic attenuation. IgG specific for budgerigars was positive. Although his respiratory symptoms were resolved without corticosteroid therapy, he developed severe dyspnea soon after the discharge to his home. The results of bronchial alveolar lavage fluid obtained at the initial admission and after the provocation challenge showed elevation of lymphocytes (34%) and eosinophils (37%). We finally diagnosed him with non-fibrotic bird-related hypersensitivity pneumonitis. His condition and HRCT findings were improved by corticosteroid treatment. All his birds were given away. He has not experienced any recurrence or deterioration of respiratory function even after withdrawal of corticosteroid.

IL-27 Levels in Bronchoalveolar Lavage Fluid in Children with Post-infectious Bronchiolitis Obliterans.

Background: Pulmonary neutrophils may play a crucial role in the development of bronchiolitis obliterans (BO) following measles virus infection. IL-27 could potentially have a negative regulatory effect on the release of reactive oxygen species and cytotoxic granules in neutrophils. Objective: To investigate the levels of IL-27 in the bronchoalveolar lavage fluid (BALF) of children with post-infectious bronchiolitis obliterans (PIBO) and analyze the relationship between IL-27 levels and neutrophil proportions. Methods: A total of 24 children with PIBO were recruited for the experimental group, while 23 children with bronchial foreign bodies were included in the control group. Bronchoscopic alveolar lavage was performed in both groups. The levels of IL-27 in BALF were measured using enzyme-linked immunosorbent assay (ELISA). The proportions of neutrophils in BALF were determined by smear staining. The relationship between the levels of IL-27 in BALF and the neutrophil proportions was analyzed by the Pearson test. Results: The levels of IL-27 in BALF were significantly lower in children with PIBO compared to children with bronchial foreign bodies (p<0.05). Additionally, the proportions of neutrophils in BALF were significantly higher in children with PIBO compared to children with bronchial foreign bodies (p<0.05). The levels of IL-27 were negatively correlated with the neutrophil proportions in BALF in children with PIBO (p<0.05), but not in children with bronchial foreign bodies (p>0.05). Conclusion: The present study suggests that a decrease in IL-27 may be associated with an increase in neutrophils in BALF and may contribute to the pathogenesis of PIBO.