Aberrant immune programming in neutrophils in cystic fibrosis.

Cystic fibrosis is a life-shortening genetic disorder, caused by mutations in the gene that encodes cystic fibrosis transmembrane-conductance regulator, a cAMP-activated chloride and bicarbonate channel. Persistent neutrophilic inflammation is a major contributor to cystic fibrosis lung disease. However, how cystic fibrosis transmembrane-conductance regulator loss of function leads to excessive inflammation and its clinical sequela remains incompletely understood. In this study, neutrophils from F508del-CF and healthy control participants were compared for gene transcription. We found that cystic fibrosis circulating neutrophils have a prematurely primed basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition. Such an irregular basal state appeared not related to the blood environment and was also observed in neutrophils derived from the F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. Lipopolysaccharides (LPS) stimulation drastically shifted the transcriptional landscape of healthy control neutrophils toward a robust immune response; however, cystic fibrosis neutrophils were immune-exhausted, reflected by abnormal cell aging and fate determination in gene programming. Moreover, cystic fibrosis sputum neutrophils differed significantly from cystic fibrosis circulating neutrophils in gene transcription with increased inflammatory response, aging, apoptosis, and necrosis, suggesting additional environmental influences on the neutrophils in cystic fibrosis lungs. Taken together, our data indicate that loss of cystic fibrosis transmembrane-conductance regulator function has intrinsic effects on neutrophil immune programming, leading to premature priming and dysregulated response to challenge.

Current Intensive Care Management of Thrombotic Thrombocytopenic Purpura: A Case Report and Updated Literature Review.

Thrombotic thrombocytopenic purpura (TTP), caused by severely reduced activity of the von Willebrand factor-cleaving protease ADAMTS13, is a medical emergency with life-threatening complications and a 90% mortality rate if left untreated. It presents a diagnostic challenge given the multiorgan involvement of the cardiovascular, gastrointestinal, and central nervous systems. Furthermore, the well-known full pentad of fever, hemolytic anemia, bleeding associated with thrombocytopenia, neurological signs, and renal disease is often absent in patients with TTP. We present a 51-year-old male adult with TTP. We utilized the PLASMIC scoring system, which predicts the likelihood of ADAMST13 activity in adults with features of thrombotic microangiopathy and thrombocytopenia with high sensitivity and specificity. We further review the literature supporting the expert statement on ICU management of patients with TTP that plasma exchange (PEX) should be initiated within 6 hours of diagnosis with adjunctive glucocorticoids, rituximab, and caplacizumab. If PEX is unavailable, plasma infusion can be started while the patient awaits transfer to a center with PEX capabilities.

ABERRANT IMMUNE PROGRAMMING IN NEUTROPHILS IN CYSTIC FIBROSIS.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the gene that encodes Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a cAMP-activated chloride and bicarbonate channel. Although multiple organ systems can be affected, CF lung disease claims the most morbidity and mortality due to chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite the clear predominance of neutrophils in these pathologies, how CFTR loss-of-function affects these cells per se remains incompletely understood. Here, we report the profiling and comparing of transcriptional signatures of peripheral blood neutrophils from CF participants and healthy human controls (HC) at the single-cell level. Circulating CF neutrophils had an aberrant basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition, suggesting that CF neutrophils in blood are prematurely primed. Such an abnormal basal state was also observed in neutrophils derived from an F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. LPS stimulation drastically shifted the transcriptional landscape of HC circulating neutrophils towards a robust immune response, however, CF neutrophils were immune-exhausted. Moreover, CF blood neutrophils differed significantly from CF sputum neutrophils in gene programming with respect to neutrophil activation and aging, as well as inflammatory signaling, highlighting additional environmental influences on the neutrophils in CF lungs. Taken together, loss of CFTR function has intrinsic effects on neutrophil immune programming that leads to premature priming and dysregulated response to challenge.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples.

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

Inflammatory pseudotumor mimicking chronic pulmonary embolism or pulmonary artery sarcoma: Report of five cases.

Inflammatory pseudotumor (IPT), also known as plasma cell granuloma, is a rare lesion of unknown etiology that occurs in many organs, especially in the lung. Here we report five cases of IPT arising in pulmonary artery mimicking chronic thromboembolic disease, not previously documented in the literature. Those cases were identified at our institute among over 2500 pulmonary endarterectomy (PEA) specimens acquired from 2000 to 2017. The cohort included three men and two women with a median age of 41 years (range: 23-54). All patients presented with dyspnea and radiologic findings of pulmonary artery thromboembolism, some concerning for intimal sarcoma. The duration between disease onset and PEA ranged from 6 months to approximately 3 years. Histologically, all cases showed proliferation of spindle cells with marked inflammatory infiltrates composed predominantly of plasma cells, histiocytes, and small lymphocytes. Ancillary studies were performed in each case and ruled out other possibilities, such as sarcoma, lymphoma, plasmacytoma, IgG4-related disease, and infection. IPT arising in pulmonary artery presenting clinically as acute or chronic thromboembolic disease is very unusual, in which clinical data, radiographic findings, and histopathologic features have to be integrated for reaching the proper diagnosis.

Inflammatory phenotype modulation in the respiratory tract and systemic circulation of e-cigarette users: a pilot study.

Over 40 million people use e-cigarettes worldwide, but the impact of chronic e-cigarette use on health has not been adequately defined. In particular, effects of e-cigarette aerosol inhalation on inflammation and host defenses across the body are not fully understood. We conducted a longitudinal cohort pilot study to explore changes in the inflammatory state and monocyte function of e-cigarette users (n = 20) versus healthy controls (n = 13) and to evaluate effects of e-cigarette use reduction on the same. Saliva, sputum, and blood were obtained from e-cigarette users at baseline and after a 2-wk intervention of decreased e-cigarette use. Overall, across 38 proteins quantified by multiplex, airway samples from e-cigarette users tended to have decreased levels of immunomodulatory proteins relative to healthy controls, whereas levels of cytokines, chemokines, and growth factors in the circulation tended to be elevated. Specifically, e-cigarette users had lower levels of IL-1 receptor antagonist (IL-1Ra) in saliva (P < 0.0001), with higher IL-1Ra and growth-regulated oncogene (GRO) levels in sputum (P < 0.01 and P < 0.05, respectively), and higher levels of both TNFß (P < 0.0001) and VEGF (P < 0.0001) in plasma. Circulating monocytes from e-cigarette users had alterations in their inflammatory phenotype in response to reduced e-cigarette use, with blunted IL-8 and IL-6 release upon challenge with bacterial lipopolysaccharide (P < 0.001 and P < 0.05, respectively), suggesting a decreased ability to appropriately respond to bacterial infection. Based on these findings, chronic inhalation of e-cigarette aerosols alters the inflammatory state of the airways and systemic circulation, raising concern for the development of both inflammatory and infectious diseases in chronic users of e-cigarettes.

An International Virtual COVID-19 Critical Care Training Forum for Healthcare Workers.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic resulted in redeployment of non-critical care-trained providers to intensive care units across the world. Concurrently, traditional venues for delivery of medical education faced major disruptions. The need for a virtual forum to fill knowledge gaps for healthcare workers caring for patients with coronavirus disease (COVID-19) was apparent in the early stages of the pandemic. Objective: The weekly, open-access COVID-19 Critical Care Training Forum (CCCTF) organized by the American Thoracic Society (ATS) provided a global audience access to timely content relevant to their learning needs. The goals of the forum were threefold: to aid healthcare providers in assessment and treatment of patients with COVID-19, to reduce provider anxiety, and to disseminate best practices. Methods: The first 13 ATS CCCTF sessions streamed live from April to July 2020. Structured debriefs followed each session and participant feedback was evaluated in planning of subsequent sessions. A second set of 14 sessions streamed from August to November 2020. Content experts were recruited from academic institutions across the United States. Results: As of July 2020, the ATS CCCTF had 2,494 live participants and 7,687 downloads for a total of 10,181 views. The majority of participants had both completed training (58.6%) and trained in critical care (53.8%). Physicians made up a majority (82.2%) of the audience that spanned the globe (61% were international attendees). Conclusion: We describe the rapid and successful implementation of an open-access medical education forum to address training and knowledge gaps among healthcare personnel caring for patients with COVID-19.

Liposome-mediated detection of SARS-CoV-2 RNA-positive extracellular vesicles in plasma.

Plasma SARS-CoV-2 RNA may represent a viable diagnostic alternative to respiratory RNA levels, which rapidly decline after infection. Quantitative PCR with reverse transcription (RT-qPCR) reference assays exhibit poor performance with plasma, probably reflecting the dilution and degradation of viral RNA released into the circulation, but these issues could be addressed by analysing viral RNA packaged into extracellular vesicles. Here we describe an assay approach in which extracellular vesicles directly captured from plasma are fused with reagent-loaded liposomes to sensitively amplify and detect a SARS-CoV-2 gene target. This approach accurately identified patients with COVID-19, including challenging cases missed by RT-qPCR. SARS-CoV-2-positive extracellular vesicles were detected at day 1 post-infection, and plateaued from day 6 to the day 28 endpoint in a non-human primate model, while signal durations for 20-60 days were observed in young children. This nanotechnology approach uses a non-infectious sample and extends virus detection windows, offering a tool to support COVID-19 diagnosis in patients without SARS-CoV-2 RNA detectable in the respiratory tract.

Chronic E-Cigarette Aerosol Inhalation Alters the Immune State of the Lungs and Increases ACE2 Expression, Raising Concern for Altered Response and Susceptibility to SARS-CoV-2.

Conventional smoking is known to both increase susceptibility to infection and drive inflammation within the lungs. Recently, smokers have been found to be at higher risk of developing severe forms of coronavirus disease 2019 (COVID-19). E-cigarette aerosol inhalation (vaping) has been associated with several inflammatory lung disorders, including the recent e-cigarette or vaping product use-associated lung injury (EVALI) epidemic, and recent studies have suggested that vaping alters host susceptibility to pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To assess the impact of vaping on lung inflammatory pathways, including the angiotensin-converting enzyme 2 (ACE2) receptor known to be involved in SARS-CoV-2 infection, mice were exposed to e-cigarette aerosols for 60 min daily for 1-6 months and underwent gene expression analysis. Hierarchical clustering revealed extensive gene expression changes occurred in the lungs of both inbred C57BL/6 mice and outbred CD1 mice, with 2,933 gene expression changes in C57BL/6 mice, and 2,818 gene expression changes in CD1 mice (>abs 1.25-fold change). Particularly, large reductions in IgA and CD4 were identified, indicating impairment of host responses to pathogens via reductions in immunoglobulins and CD4 T cells. CD177, facmr, tlr9, fcgr1, and ccr2 were also reduced, consistent with diminished host defenses via decreased neutrophils and/or monocytes in the lungs. Gene set enrichment (GSE) plots demonstrated upregulation of gene expression related to cell activation specifically in neutrophils. As neutrophils are a potential driver of acute lung injury in COVID-19, increased neutrophil activation in the lungs suggests that vapers are at higher risk of developing more severe forms of COVID-19. The receptor through which SARS-CoV-2 infects host cells, ACE2, was found to have moderate upregulation in mice exposed to unflavored vape pens, and further upregulation (six-fold) with JUUL mint aerosol exposure. No changes were found in mice exposed to unflavored Mod device-generated aerosols. These findings suggest that specific vaping devices and components of e-liquids have an effect on ACE2 expression, thus potentially increasing susceptibility to SARS-CoV-2. In addition, exposure to e-cigarette aerosols both with and without nicotine led to alterations in eicosanoid lipid profiles within the BAL. These data demonstrate that chronic, daily inhalation of e-cigarette aerosols fundamentally alters the inflammatory and immune state of the lungs. Thus, e-cigarette vapers may be at higher risk of developing infections and inflammatory disorders of the lungs.

Compositional Differences in the Oral Microbiome of E-cigarette Users.

Electronic (e)-cigarettes have been advocated as a safer alternative to conventional tobacco cigarettes. However, there is a paucity of data regarding the impact of e-cigarette aerosol deposition on the human oral microbiome, a key component in human health and disease. We aimed to fill this knowledge gap through a comparative analysis of the microbial community profiles from e-cigarette users and healthy controls [non-smokers/non-vapers (NSNV)]. Moreover, we sought to determine whether e-cigarette aerosol exposure from vaping induces persistent changes in the oral microbiome. To accomplish this, salivary and buccal mucosa samples were collected from e-cigarette users and NSNV controls, with additional oral samples collected from e-cigarette users after 2 weeks of decreased use. Total DNA was extracted from all samples and subjected to PCR amplification and sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Our analysis revealed several prominent differences associated with vaping, specific to the sample type (i.e., saliva and buccal). In the saliva, e-cigarette users had a significantly higher alpha diversity, observed operational taxonomic units (OTUs) and Faith's phylogenetic diversity (PD) compared to NSNV controls, which declined with decreased vaping. The buccal mucosa swab samples were marked by a significant shift in beta diversity between e-cigarette users and NSNV controls. There were also significant differences in the relative abundance of several bacterial taxa, with a significant increase in Veillonella and Haemophilus in e-cigarette users. In addition, nasal swabs demonstrated a trend toward higher colonization rates with Staphylococcus aureus in e-cigarette users relative to controls (19 vs. 7.1%; p = n.s.). Overall, these data reveal several notable differences in the oral bacterial community composition and diversity in e-cigarette users as compared to NSNV controls.

Mucosal Immunity in Cystic Fibrosis.

The highly complex and variable genotype-phenotype relationships observed in cystic fibrosis (CF) have been an area of growing interest since the discovery of the CF transmembrane conductance regulator (CFTR) gene >30 y ago. The consistently observed excessive, yet ineffective, activation of both the innate and adaptive host immune systems and the establishment of chronic infections within the lung, leading to destruction and functional decline, remain the primary causes of morbidity and mortality in CF. The fact that both inflammation and pathogenic bacteria persist despite the introduction of modulator therapies targeting the defective protein, CFTR, highlights that we still have much to discover regarding mucosal immunity determinants in CF. Gene modifier studies have overwhelmingly implicated immune genes in the pulmonary phenotype of the disease. In this context, we aim to review recent advances in our understanding of the innate and adaptive immune systems in CF lung disease.

Metabolic Syndrome and COVID-19 Mortality Among Adult Black Patients in New Orleans.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) mortality is high in patients with hypertension, obesity, and diabetes. We examined the association between hypertension, obesity, and diabetes, individually and clustered as metabolic syndrome (MetS), and COVID-19 outcomes in patients hospitalized in New Orleans during the peak of the outbreak. RESEARCH DESIGN AND METHODS: Data were collected from 287 consecutive patients with COVID-19 hospitalized at two hospitals in New Orleans, LA from 30 March to 5 April 2020. MetS was identified per World Health Organization criteria. RESULTS: Among 287 patients (mean age 61.5 years; female, 56.8%; non-Hispanic black, 85.4%), MetS was present in 188 (66%). MetS was significantly associated with mortality (adjusted odds ratio [aOR] 3.42 [95% CI 1.52-7.69]), intensive care unit (ICU) (aOR 4.59 [CI 2.53-8.32]), invasive mechanical ventilation (IMV) (aOR 4.71 [CI 2.50-8.87]), and acute respiratory distress syndrome (ARDS) (aOR 4.70 [CI 2.25-9.82]) compared with non-MetS. Multivariable analyses of hypertension, obesity, and diabetes individually showed no association with mortality. Obesity was associated with ICU (aOR 2.18 [CI, 1.25-3.81]), ARDS (aOR 2.44 [CI 1.28-4.65]), and IMV (aOR 2.36 [CI 1.33-4.21]). Diabetes was associated with ICU (aOR 2.22 [CI 1.24-3.98]) and IMV (aOR 2.12 [CI 1.16-3.89]). Hypertension was not significantly associated with any outcome. Inflammatory biomarkers associated with MetS, CRP, and lactate dehydrogenase (LDH) were associated with mortality (CRP [aOR 3.66] [CI 1.22-10.97] and LDH [aOR 3.49] [CI 1.78-6.83]). CONCLUSIONS: In predominantly black patients hospitalized for COVID-19, the clustering of hypertension, obesity, and diabetes as MetS increased the odds of mortality compared with these comorbidities individually.

BMI is Associated with Coronavirus Disease 2019 Intensive Care Unit Admission in African Americans.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has disproportionately impacted the African American community. This study aims to identify the risk factors for severe COVID-19 disease in African American patients. METHODS: This was a retrospective cross-sectional analysis of African American patients with COVID-19 treated between March 12 and April 9, 2020, at a single tertiary center. The primary outcome of interest was severe disease defined as those requiring intensive care unit (ICU) admission. RESULTS: The study included 158 consecutive patients. The mean age was 57 years, and 61% were women. The mean (SD) of BMI was 33.2 (8.6) kg/m2 . Overall, patients admitted to the ICU were older (62 vs. 55 years, P = 0.003) and had higher BMI (36.5 kg/m2 vs. 31.9 kg/m2 , P = 0.002). In unadjusted and adjusted analysis, the factors most associated with ICU admission in this sample were age (adjusted odds ratio [aOR]: 1.073; 95% CI: 1.033-1.114), BMI (aOR: 1.115; 95% CI: 1.052-1.182), and lung disease (aOR: 3.097; 95% CI: 1.137-8.437). CONCLUSIONS: This study identified risk factors for severe disease in COVID-19, specifically in an African American population. Further inclusive research aimed at optimizing clinical care relevant to the African American population is critical to ensure an equitable response to COVID-19.

E-cigarette use increases susceptibility to bacterial infection by impairment of human neutrophil chemotaxis, phagocytosis, and NET formation.

E-cigarettes are portrayed as safer relative to conventional tobacco. However, burgeoning evidence suggests that E-cigarettes may adversely affect host defenses. However, the precise mechanisms by which E-cigarette vapor alters innate immune cell function have not been fully elucidated. We determined the effects of E-cigarette exposure on the function and responses to infectious challenge of the most abundant innate immune cell, the neutrophil, using isolated human neutrophils and a mouse model of gram-negative infection. Our results revealed that human neutrophils exposed to E-cigarette vapor had 4.2-fold reductions in chemotaxis toward the bacterial cell-well component f-Met-Leu-Phe (P < 0.001). F-actin polarization and membrane fluidity were also adversely affected by E-cigarette vapor exposure. E-cigarette-exposed human neutrophils exhibited a 48% reduction in production of reactive oxygen species (ROS; P < 0.001). Given the central role of ROS in neutrophil extracellular trap (NET) production, NET production was quantified, and E-cigarette vapor exposure was found to reduce NETosis by 3.5-fold (P < 0.01); formulations with and without nicotine containing propylene glycol exhibiting significant suppressive effects. However, noncanonical NETosis was unaffected. In addition, exposure to E-cigarette vapor lowered the rate of phagocytosis of bacterial bioparticles by 47% (P < 0.05). In our physiological mouse model of chronic E-cigarette exposure and sepsis, E-cigarette vapor inhalation led to reduced neutrophil migration in infected spaces and a higher burden of Pseudomonas. These findings provide evidence that E-cigarette use adversely impacts the innate immune system and may place E-cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.

Effects of Chronic Inhalation of Electronic Cigarette Vapor Containing Nicotine on Neurotransmitters in the Frontal Cortex and Striatum of C57BL/6 Mice.

Electronic (E)-cigarettes are the latest form of nicotine delivery device and are highly popular in the general population. It is currently unknown whether vaping E-cigarettes (E-CIGs) leads to nicotine addiction. Alterations in the levels of the neurotransmitters in the mesocorticolimbic areas have been reported to mediate the initiation and development of nicotine addiction. Therefore, to determine whether E-CIGs activate the same addiction pathways as conventional cigarettes, we investigated for the effects of daily inhalation of nicotine (24 mg/ml)-containing E-CIG vapor for 6 months on the concentrations of these neurotransmitters in the frontal cortex (FC) and striatum (STR) of male C57BL/6 mice as compared to control group that was exposed to air only. We reported here that 6-month E-CIG vapor containing nicotine inhalation decreased dopamine concentration only in the STR. There were no changes in serotonin concentrations in the FC or STR. Chronic E-CIG exposure also increased glutamate concentration in the STR alone, while glutamine concentrations were increased in both the FC and STR. We found that E-CIG exposure also decreased GABA concentration only in the FC. These data suggest that chronic E-CIG use alters homeostasis of several neurotransmitters in the mesocorticolimbic areas, which may result in the development of nicotine dependence in E-CIG users.