Is there a role for lung or bronchial biopsies for the diagnosis of mycobacterial pulmonary disease in patients with bronchiectasis?

Background: Workup of bronchiectasis patients mandates microbiological characterization often being sought via Bronchoscopy. However, whether to perform bronchial or lung biopsies, is unknown, especially for the diagnosis of NTM pulmonary disease. We aimed to assess the current practice and yield of the different bronchoscopic procedures in this setting. Methods: Data from an adult cohort with bronchiectasis referred for bronchoscopy for microbiologic sampling was reviewed, including demographics, etiology, imaging and results of the different bronchoscopic procedures performed. Results: 127 subjects were analyzed (mean age 61, 56% female). BAL culture was positive in 44%. Frequent pathogens were Hemophilus Influenza (20%), pseudomonas aeruginosa (8%) and Staphylococcus aureus (7%). NTM and tuberculosis were found in 6% and 1.5% respectively. BAL cytology was sent in 125 procedures, EBB was performed in 51 patients (40%) and TBLB in 38 patients (30%). BAL cytology and both EBB and TBB (including tissue cultures) had no benefit over BAL with respect to microbiological diagnosis, including identification of mycobacterial disease. Conclusions: In adult subjects with Non-CF bronchiectasis requiring bronchoscopy for microbiological characterization, BAL cytology and lung tissue biopsies were frequently performed but were of minimal additional benefit over BAL culture (including for mycobacterial pulmonary disease), and are most likely futile.

Breath of Health: spectroscopy-based breath test for the detection of SARS-CoV-2.

BACKGROUND: Nucleic acid amplification tests (NAAT) are considered the gold standard for COVID-19 diagnosis. These tests require professional manpower and equipment, long processing and swab sampling which is unpleasant to the patients. Several volatile organic compounds (VOCs) have been identified in the breath of COVID-19 patients. Detection of these VOCs using a breath test could help rapidly identify COVID-19 patients. OBJECTIVE: Assess the accuracy of 'Breath of Health' (BOH) COVID-19 Fourier-transform infra-red (FTIR) Spectroscopy-based breath test. METHODS: Breath samples from patients with or without symptoms suggestive for COVID-19 who had NAAT results were collected using Tedlar bags and were blindly analysed using BOH FTIR spectroscopy. BOH Measures several VOCs simultaneously and differentiating positive and negative results. BOH results were compared to NAAT results as gold standard. RESULTS: Breath samples from 531 patients were analysed. The sensitivity of BOH breath test was found to be 79.5% and specificity was 87.2%. Positive predictive value (PPV) was 74.7% and negative predictive value (NPV) 90.0%. Calculated accuracy rate was 84.8% and area under the curve 0.834. Subgroup analysis revealed that the NPV of patients without respiratory symptoms was superior over the NPV of symptomatic patients (94.7% vs 80.7%, P-value < 0.0001) and PPV of patients with respiratory symptoms outranks the PPV of individuals without symptoms (85.3% vs 69.2%, P-value 0.0196). CONCLUSION: We found BOH COVID-19 breath test to be a patient-friendly, rapid, non-invasive diagnostic test with high accuracy rate and NPV that could efficiently rule out COVID-19 especially among individuals with low pre-test probability.

RNA Sequencing Reveals Unique Transcriptomic Signatures of the Thyroid in a Murine Lung Cancer Model Treated with PD-1 and PD-L1 Antibodies.

Immune checkpoint inhibitors (ICI) are commonly associated with thyroid immune-related adverse events, yet the mechanism has not been fully elucidated. We aimed to further explore the mechanism of ICI-induced thyroid dysfunction by assessing changes induced in the thyroid transcriptome by ICI treatment (αPD-1/αPD-L1) in a lung cancer murine model. RNA-sequencing of thyroid tissues revealed 952 differentially expressed genes (DEGs) with αPD-1 treatment (|fold-change| ≥1.8, FDR < 0.05). Only 35 DEG were identified with αPD-L1, and we therefore focused on the αPD-1 group alone. Ingenuity Pathway Analysis revealed that of 952 DEGs with αPD-1 treatment, 362 were associated with functions of cell death and survival, with predicated activation of pathways for apoptosis and necrosis (Z = 2.89 and Z = 3.21, respectively) and negative activation of pathways for cell viability and cell survival (Z = -6.22 and Z = -6.45, respectively). Compared to previously published datasets of interleukin-1ß and interferon γ-treated human thyroid cells, apoptosis pathways were similarly activated. However, unique changes related to organ inflammation and upstream regulation by cytokines were observed. Our data suggest that there are unique changes in gene expression in the thyroid associated with αPD-1 therapy. ICI-induced thyroid dysfunction may be mediated by increased tissue apoptosis resulting in destructive thyroiditis.

Elevated cfDNA after exercise is derived primarily from mature polymorphonuclear neutrophils, with a minor contribution of cardiomyocytes.

Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown. Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, ß-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level. We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage.

The DNA methylome of human vascular endothelium and its use in liquid biopsies.

BACKGROUND: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. METHODS: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. FINDINGS: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. CONCLUSIONS: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. FUNDING: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.

How Accurate Is the Diagnosis of "Chronic Obstructive Pulmonary Disease" in Patients Hospitalized with an Acute Exacerbation?

Rationale: COPD diagnosis requires relevant symptoms and an FEV1/FVC ratio of <0.7 post-bronchodilator on spirometry. Patients are frequently labeled as COPD based on clinical presentation and admitted to the hospital with this diagnosis even though spirometry is either not available or has never been performed. The aim of this study was to evaluate the accuracy of COPD diagnosis based on post-bronchodilator spirometry, following hospital admission for COPD exacerbation. Methods: This is a retrospective study with a cross-sectional analysis of a subgroup of patients. Demographic and clinical data and pre-admission spirometry were collected from electronic records of patients hospitalized with a primary diagnosis of COPD. Patients without available spirometry were contacted for a pulmonary consultation and spirometry. Three groups were compared: patients with a confirmed COPD diagnosis (FEV1/FVC < 0.7), without COPD (FEV1/FVC > 0.7), and those who have never performed spirometry. Results: A total of 1138 patients with a recorded diagnosis of COPD were identified of which 233 patients were included in the analysis. Only 44.6% of patients had confirmed COPD according to GOLD criteria. In total, 32.6% of the patients had never undergone spirometry but were treated as COPD, and 22.7% had performed spirometry without evidence of COPD. Recurrent admission due to COPD was a strong predictor of a confirmed COPD diagnosis. Conclusions: Among the patients admitted to the hospital with a COPD diagnosis, a high proportion were not confirmed by the current GOLD report or had never performed spirometry. Stricter implementation of the diagnostic criteria of COPD in admitted patients is necessary to improve diagnosis and the treatment outcomes in these patients.

Targeted nanoparticles modify neutrophil function in vivo.

Neutrophils play critical roles in a broad spectrum of clinical conditions. Accordingly, manipulation of neutrophil function may provide a powerful immunotherapeutic approach. However, due to neutrophils characteristic short half-life and their large population number, this possibility was considered impractical. Here we describe the identification of peptides which specifically bind either murine or human neutrophils. Although the murine and human neutrophil-specific peptides are not cross-reactive, we identified CD177 as the neutrophil-expressed binding partner in both species. Decorating nanoparticles with a neutrophil-specific peptide confers neutrophil specificity and these neutrophil-specific nanoparticles accumulate in sites of inflammation. Significantly, we demonstrate that encapsulating neutrophil modifying small molecules within these nanoparticles yields specific modulation of neutrophil function (ROS production, degranulation, polarization), intracellular signaling and longevity both in vitro and in vivo. Collectively, our findings demonstrate that neutrophil specific targeting may serve as a novel mode of immunotherapy in disease.

Recurrent haemoptysis: a rare diagnosis of kaposiform lymphangiomatosis and review of literature.

Kaposiform lymphangiomatosis (KLA) is a rare clinicopathological entity among lymphatic anomalies. The main involved sites are the mediastinum and the lungs but the disease can also affect multiple extrathoracic organs. Little is known about the pathophysiology, the natural history, the treatment response and the long-term outcome of this disorder. KLA is typically diagnosed in childhood. We present here the case of an adult man with 13 years recurrent episodes of haemoptysis who was finally found to suffer from KLA. Following this, we present a comprehensive review of the literature.

Neutrophil phenotypes and functions in cancer: A consensus statement.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies.

BACKGROUND: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. METHODS: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. RESULTS: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. CONCLUSIONS: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

Residual symptoms, lung function, and imaging findings in patients recovering from SARS-CoV-2 infection.

Symptoms following acute COVID-19 infection are common, but their relationship to initial COVID-19 severity is unclear. We hypothesize that residual symptoms are related to disease severity, and severe acute COVID-19 infection is more likely to cause residual pulmonary damage. This study aims to evaluate symptoms, lung function, and abnormal imaging within 3 months following COVID-19 infection, and to determine whether they are related to initial disease severity. A cross-sectional study was carried out at a designated post-COVID clinic in Hadassah Medical Center, Jerusalem, Israel. Patients with PCR-confirmed SARS-CoV-2 infection were evaluated within 12 weeks following infection and included both admitted and non-admitted subjects. All study participants underwent assessment of symptoms, quality of life (SGRQ), pulmonary function tests, and imaging. A total of 208 patients (age 49.3 ± 16 years) were included in the study. Initial disease severity was mild in 86, moderate in 49, and severe in 73 patients. At the time of follow-up, there were no differences in frequency of residual symptoms or in SGRQ score between groups. Patients with severe COVID-19 were more likely to have residual dyspnea (p = 0.04), lower oxygen saturation (p < 0.01), lower FVC and TLC (p < 0.001, p = 0.03 respectively), abnormal CXR (p < 0.01), and abnormal CT scan (p < 0.01) compared to other groups.Frequency of symptoms and impairment of quality of life at 12 week follow-up are common and are not related to severity of initial COVID-19 disease. In contrast, reduced lung function and abnormal pulmonary imaging are more common in patients with more severe acute COVID-19 infection.

The Bilateral Interplay between Cancer Immunotherapies and Neutrophils' Phenotypes and Sub-Populations.

Immunotherapy has become a leading modality for the treatment of cancer, but despite its increasing success, a substantial number of patients do not benefit from it. Cancer-related neutrophils have become, in recent years, a subject of growing interest. Distinct sub-populations of neutrophils have been identified at advanced stages of cancer. In this study, we aimed to evaluate the role of neutrophils in mediating the efficacy of immune checkpoint inhibitors (ICI) treatments (α-PD-1/PD-L1), by assessing lung tumor models in mice. We found that G-CSF overexpression by the tumor significantly potentiates the efficacy of ICI, whereas neutrophils' depletion abrogated their responses. Adoptive transfer of circulating normal-density neutrophils (NDN) resulted in significantly reduced tumor growth, whereas low-density neutrophils (LDN) had no effect. We next investigated the effect of ICI on neutrophils' functions. Following α-PD-L1 treatment, NDN displayed increased ROS production and increased cytotoxicity toward tumor cells but decreased degranulation. Together, our results suggest that neutrophils are important mediators of the ICI treatments and that mainly NDN are modulated following α-PD-L1 treatment. This research provides a better understanding of the function of neutrophils following immunotherapies and their impact on the efficacy of immunotherapy, supporting better understanding and future improvement of currently available treatments.

Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils.

Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils' plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.

Tumor-Associated Neutrophils Drive B-cell Recruitment and Their Differentiation to Plasma Cells.

A major mechanism through which neutrophils have been suggested to modulate tumor progression involves the interaction and subsequent modulation of other infiltrating immune cells. B cells have been found to infiltrate various cancer types and play a role in tumor immunity, offering new immunotherapy opportunities. Nevertheless, the specific impact of tumor-associated neutrophils (TAN) on B cells has largely been overlooked. In the current study, we aimed to characterize the role of TANs in the recruitment and modulation of B cells in the tumor microenvironment (TME). We showed that TANs actively participate in the recruitment of B cells to the TME and identified TNFα as the major cytokine mediating B-cell chemotaxis by TANs. The recruitment of CD45+B220+CD138- splenic B cells by TANs in vitro resulted in B-cell phenotypic modulation, with 68.6% ± 2.1% of the total migrated B cells displaying a CD45-B220+CD138+ phenotype, which is typical for plasma cells. This phenotype mirrored the large proportion (54.0% ± 6.1%) of CD45-B220+CD138+ intratumoral B cells (i.e., plasma cells) in Lewis lung carcinoma tumors. We next confirmed that the differentiation of CD45+B220+CD138- B cells to functionally active CD45-B220+CD138+ plasma cells required contact with TANs, was independent of T cells, and resulted in IgG production. We further identified membranal B-cell activating factor (BAFF) on TANs as a potential contact mechanism mediating B-cell differentiation, as blocking BAFF-receptor (BAFF-R) significantly reduced IgG production by 20%. Our study, therefore, demonstrates that TANs drive the recruitment and modulation of B cells into plasma cells in the TME, hence opening new avenues in the targeting of the immune system in cancer.

Therapeutic Success of Tiotropium/Olodaterol, Measured Using the Clinical COPD Questionnaire (CCQ), in Routine Clinical Practice: A Multinational Non-Interventional Study.

BACKGROUND: The Clinical COPD Questionnaire (CCQ) is a simple patient-reported tool to measure clinical control of chronic obstructive pulmonary disease (COPD). OBJECTIVE: This open-label, single-arm, non-interventional study (NCT03663569) investigated changes in CCQ score during treatment with tiotropium/olodaterol in clinical practice. METHODS: Data were included from consenting COPD patients, enrolled in Bulgaria, Czech Republic, Hungary, Israel, Lithuania, Poland, Romania, Russia, Slovenia, Switzerland and Ukraine, who were receiving a new prescription for tiotropium/olodaterol according to the treating physician in a real-world environment. The primary endpoint was the occurrence of therapeutic success, defined as a 0.4-point decrease in CCQ score after treatment with tiotropium/olodaterol for approximately 6 weeks. RESULTS: Overall, 4819 patients were treated; baseline and Week 6 CCQ scores were available for 4700 patients, mostly classified as Global Initiative for Chronic Obstructive Lung Disease (GOLD) B (51.6%) or D (42.7%). After 6 weeks' treatment, 81.4% (95% confidence interval [95% CI] 80.24-82.49) of patients achieved therapeutic success; mean improvement in overall CCQ score was 1.02 points (95% CI 1.00-1.05). Improved CCQ score was seen in 92.2% of patients (95% CI 91.43-92.98), 2.5% had no change and 5.3% showed a worsening. When stratified by prior treatment, the greatest benefit was seen in treatment-naïve patients, with 85.7% achieving therapeutic success, compared with 79.5% of those pretreated with long-acting ß2-agonist (LABA)/inhaled corticosteroid (ICS) and 74.2% of those pretreated with LABA or long-acting muscarinic antagonist (LAMA) monotherapy. Overall, rescue medication decreased by 1.25 puffs/day (95% CI 1.19-1.31) versus baseline. In total, 29 patients (0.6%) reported drug-related adverse events and 7 patients reported serious adverse events (0.15%). CONCLUSION: In 4700 COPD patients, 6 weeks' treatment with tiotropium/olodaterol, as initial treatment or follow-up to LAMA or LABA monotherapy or LABA/ICS, improved CCQ and decreased rescue medication use. The adverse event profile was consistent with the known safety profile of tiotropium/olodaterol.

Blocking Migration of Polymorphonuclear Myeloid-Derived Suppressor Cells Inhibits Mouse Melanoma Progression.

BACKGROUND: Despite recent improvement in the treatment of malignant melanoma by immune-checkpoint inhibitors, the disease can progress due to an immunosuppressive tumor microenvironment (TME) mainly represented by myeloid-derived suppressor cells (MDSC). However, the relative contribution of the polymorphonuclear (PMN) and monocytic (M) MDSC subsets to melanoma progression is not clear. Here, we compared both subsets regarding their immunosuppressive capacity and recruitment mechanisms. Furthermore, we inhibited PMN-MDSC migration in vivo to determine its effect on tumor progression. METHODS: Using the RET transgenic melanoma mouse model, we investigated the immunosuppressive function of MDSC subsets and chemokine receptor expression on these cells. The effect of CXCR2 inhibition on PMN-MDSC migration and tumor progression was studied in RET transgenic mice and in C57BL/6 mice after surgical resection of primary melanomas. RESULTS: Immunosuppressive capacity of intratumoral M- and PMN-MDSC was comparable in melanoma bearing mice. Anti-CXCR2 therapy prolonged survival of these mice and decreased the occurrence of distant metastasis. Furthermore, this therapy reduced the infiltration of melanoma lesions and pre-metastatic sites with PMN-MDSC that was associated with the accumulation of natural killer (NK) cells. CONCLUSIONS: We provide evidence for the tumor-promoting properties of PMN-MDSC as well as for the anti-tumor effects upon their targeting in melanoma bearing mice.