Human alveolar lining fluid from the elderly promotes Mycobacterium tuberculosis intracellular growth and translocation into the cytosol of alveolar epithelial cells.

The elderly population is highly susceptible to developing respiratory diseases, including tuberculosis, a devastating disease caused by the airborne pathogen Mycobacterium tuberculosis (M.tb) that kills one person every 18 seconds. Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF), which dictates host-cell interactions. We previously determined that age-associated dysfunction of soluble innate components in human ALF leads to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial type cells (ATs), another critical lung cellular determinant of infection. We observed that elderly ALF (E-ALF)-exposed M.tb had significantly increased intracellular growth with rapid replication in ATs compared to adult ALF (A-ALF)-exposed bacteria, as well as a dampened inflammatory response. A potential mechanism underlying this accelerated growth in ATs was our observation of increased bacterial translocation into the cytosol, a compartment that favors bacterial replication. These findings in the context of our previous studies highlight how the oxidative and dysfunctional status of the elderly lung mucosa determines susceptibility to M.tb infection, including dampening immune responses and favoring bacterial replication within alveolar resident cell populations, including ATs, the most abundant resident cell type within the alveoli.

Exposure of Mycobacterium tuberculosis to human alveolar lining fluid shows temporal and strain-specific adaptation to the lung environment.

Upon infection, Mycobacterium tuberculosis ( M.tb ) reaches the alveolar space and comes in close contact with human alveolar lining fluid (ALF) for an uncertain period of time prior to its encounter with alveolar cells. We showed that homeostatic ALF hydrolytic enzymes modify the M.tb cell envelope, driving M.tb -host cell interactions. Still, the contribution of ALF during M.tb infection is poorly understood. Here, we exposed 4 M.tb strains with different levels of virulence, transmissibility, and drug resistance (DR) to physiological concentrations of human ALF for 15-min and 12-h, and performed RNA sequencing. Gene expression analysis showed a temporal and strain-specific adaptation to human ALF. Differential expression (DE) of ALF-exposed vs. unexposed M.tb revealed a total of 397 DE genes associated with lipid metabolism, cell envelope and processes, intermediary metabolism and respiration, and regulatory proteins, among others. Most DE genes were detected at 12-h post-ALF exposure, with DR- M.tb strain W-7642 having the highest number of DE genes. Interestingly, genes from the KstR2 regulon, which controls the degradation of cholesterol C and D rings, were significantly upregulated in all strains post-ALF exposure. These results indicate that M.tb -ALF contact drives initial metabolic and physiologic changes in M.tb , with potential implications in infection outcome. IMPORTANCE: Tuberculosis, caused by airborne pathogen Mycobacterium tuberculosis ( M.tb ), is one of the leading causes of mortality worldwide. Upon infection, M.tb reaches the alveoli and gets in contact with human alveolar lining fluid (ALF), where ALF hydrolases modify the M.tb cell envelope driving subsequent M.tb -host cell interactions. Still, the contributions of ALF during infection are poorly understood. We exposed 4 M.tb strains to ALF for 15-min and 12-h and performed RNA sequencing, demonstrating a temporal and strain-specific adaptation of M.tb to ALF. Interestingly, genes associated with cholesterol degradation were highly upregulated in all strains. This study shows for the first time that ALF drives global metabolic changes in M.tb during the initial stages of the infection, with potential implications in disease outcome. Biologically relevant networks and common and strain-specific bacterial determinants derived from this study could be further investigated as potential therapeutic candidates.

A new tractable method for generating human alveolar macrophage-like cells in vitro to study lung inflammatory processes and diseases.

Alveolar macrophages (AMs) are unique lung resident cells that contact airborne pathogens and environmental particulates. The contribution of human AMs (HAMs) to pulmonary diseases remains poorly understood due to the difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. Thus, there remains an unmet need for cost-effective methods for generating and/or differentiating primary cells into a HAM phenotype, particularly important for translational and clinical studies. We developed cell culture conditions that mimic the lung alveolar environment in humans using lung lipids, that is, Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (granulocyte macrophage colony-stimulating factor, transforming growth factor-ß, and interleukin 10) that facilitate the conversion of blood-obtained monocytes to an AM-like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function and provides a readily accessible model to study HAM in infectious and inflammatory disease processes, as well as therapies and vaccines. IMPORTANCE Millions die annually from respiratory disorders. Lower respiratory track gas-exchanging alveoli maintain a precarious balance between fighting invaders and minimizing tissue damage. Key players herein are resident AMs. However, there are no easily accessible in vitro models of HAMs, presenting a huge scientific challenge. Here, we present a novel model for generating AML cells based on differentiating blood monocytes in a defined lung component cocktail. This model is non-invasive, significantly less costly than performing a bronchoalveolar lavage, yields more AML cells than HAMs per donor, and retains their phenotype in culture. We have applied this model to early studies of M. tuberculosis and SARS-CoV-2. This model will significantly advance respiratory biology research.

A new tractable method for generating Human Alveolar Macrophage Like cells in vitro to study lung inflammatory processes and diseases.

Alveolar macrophages (AMs) are unique lung resident cells that contact airborne pathogens and environmental particulates. The contribution of human AMs (HAM) to pulmonary diseases remains poorly understood due to difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. Thus, there remains an unmet need for cost-effective methods for generating and/or differentiating primary cells into a HAM phenotype, particularly important for translational and clinical studies. We developed cell culture conditions that mimic the lung alveolar environment in humans using lung lipids, i.e. , Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (GM-CSF, TGF-ß, and IL-10) that facilitate the conversion of blood-obtained monocytes to an AM-Like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function, and provides a readily accessible model to study HAM in infectious and inflammatory disease processes, as well as therapies and vaccines. IMPORTANCE: Millions die annually from respiratory disorders. Lower respiratory track gas-exchanging alveoli maintain a precarious balance between fighting invaders and minimizing tissue damage. Key players herein are resident AMs. However, there are no easily accessible in vitro models of HAMs, presenting a huge scientific challenge. Here we present a novel model for generating AML cells based on differentiating blood monocytes in a defined lung component cocktail. This model is non-invasive, significantly less costly than performing a bronchoalveolar lavage, yields more AML cells than HAMs per donor and retains their phenotype in culture. We have applied this model to early studies of M. tuberculosis and SARS-CoV-2. This model will significantly advance respiratory biology research.

A marmoset model for Mycobacterium avium complex pulmonary disease.

RATIONALE: Mycobacterium avium complex, is the most common nontuberculous mycobacterial respiratory pathogen in humans. Disease mechanisms are poorly understood due to the absence of a reliable animal model for M. avium complex pulmonary disease. OBJECTIVES: The objectives of this study were to assess the susceptibility, immunologic and histopathologic responses of the common marmoset (Callithrix jacchus) to M. avium complex pulmonary infection. METHODS: 7 adult female marmosets underwent endobronchial inoculation with 108 colony-forming units of M. intracellulare and were monitored for 30 or 60 days. Chest radiograph was assessed at baseline (prior to infection) and at the time of sacrifice (30 days for 3 animals and 60 days for 4 animals), and bronchoalveolar lavage cytokines, histopathology and cultures of the bronchoalveolar lavage, lungs, liver and kidney were assessed at time of sacrifice. Serum cytokines were monitored at baseline and weekly for 30 days for all animals and at 60 days for those alive. Group differences in serum cytokine measurements between those that tested positive versus negative for the M. intracellulare infection were assessed using a series of linear mixed models. MEASUREMENTS AND MAIN RESULTS: Five of seven animals (two at 30 days and three at 60 days of infection) had positive lung cultures for M. intracellulare. Extra-pulmonary cultures were positive in three animals. All animals appeared healthy throughout the study. All five animals with positive lung cultures had radiographic changes consistent with pneumonitis. At 30 days, those with M. intracellulare lung infection showed granulomatous inflammation, while at 60 days there were fewer inflammatory changes but bronchiectasis was noted. The cytokine response in the bronchoalveolar lavage fluid was uniformly greater in the animals with positive M. intracellulare cultures than those without a productive infection, with greater levels at 30-days compared to 60-days. Similarly, serum cytokines were more elevated in the animals that had positive M. intracellulare cultures compared to those without a productive infection, peaking 14-21 days after inoculation. CONCLUSION: Endobronchial instillation of M. intracellulare resulted in pulmonary mycobacterial infection in marmosets with a differential immune response, radiographic and histopathologic abnormalities, and an indolent course consistent with M. avium complex lung infection in humans.

The Aging Human Lung Mucosa: A Proteomics Study.

The older adult population, estimated to double by 2050, is at increased risk of respiratory infections and other pulmonary diseases. Biochemical changes in the lung alveolar lining fluid (ALF) and in alveolar compartment cells can alter local immune responses as we age, generating opportunities for invading pathogens to establish successful infections. Indeed, the lung alveolar space of older adults is a pro-inflammatory, pro-oxidative, dysregulated environment that remains understudied. We performed an exploratory, quantitative proteomic profiling of the soluble proteins present in ALF, developing insight into molecular fingerprints, pathways, and regulatory networks that characterize the alveolar space in old age, comparing it to that of younger individuals. We identified 457 proteins that were significantly differentially expressed in older adult ALF, including increased production of matrix metalloproteinases, markers of cellular senescence, antimicrobials, and proteins of neutrophilic granule origin, among others, suggesting that neutrophils in the lungs of older adults could be potential contributors to the dysregulated alveolar environment with increasing age. Finally, we describe a hypothetical regulatory network mediated by the serum response factor that could explain the neutrophilic profile observed in the older adult population.

Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.

Quality of the discussion of asthma on twitter.

IntroductionPatients obtain a large amount of medical information online. Much of this information may not be reliable or of high quality. We investigated what influences the discussion of asthma on Twitter by evaluating the most popular tweets and the quality of the links shared.MethodsWe used Symplur Signals to extract data from Twitter examining characteristics of the top 100 most shared tweets and the 50 most shared links that included the hashtag #asthma. Information on each site was assessed using an Asthma Content score, and validated DISCERN scores and HONCode criteria.ResultsThe top 100 asthma-related tweets were shared 10,169 times and had 16,044 likes. Healthcare organizations posted 49 of the top 100 tweets, non-healthcare individuals posted 20, non-healthcare organizations posted 16 and clinicians posted 14. Of the top 100 tweets, 62 were educational, 11 research-related, 10 political and 15 promotional. The top 50 links were shared 6009 times (median number of shares 92 per link (range 60-710)). Links most commonly (42%) led to educational content while 24% of links led to research articles, 22% to promotional websites, and 12% to political websites. Educational links had higher Asthma Content scores than other links (p < 0.005). Overall, all three scores were low for all types of links. Only 34% of sites met HONCode criteria, and 14% were assessed as high quality by DISCERN score.ConclusionThe top tweets using the hashtag #asthma were commonly educational. The majority of top links on Twitter scored poorly on asthma content, quality, and reliability.

Host- and age-dependent transcriptional changes in Mycobacterium tuberculosis cell envelope biosynthesis genes after exposure to human alveolar lining fluid.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis ( M . tb ), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M . tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M . tb upon contact, defining subsequent M . tb -host cell interactions and infection outcomes in vitro and in vivo . We also demonstrated that ALF from 60+ year old elders (E-ALF) vs . healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M . tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M . tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M . tb exposure to E-ALF shows lesser transcriptional response, with most of the M . tb genes unchanged or downregulated. Overall, this study indicates that M . tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

A Safety and Tolerability Study of Thin Film Freeze-Dried Tacrolimus for Local Pulmonary Drug Delivery in Human Subjects.

Due to the low and erratic bioavailability of oral tacrolimus (TAC), the long-term survival rate following lung transplantation remained low compared to other solid organs. TAC was reformulated and developed as inhaled formulations by thin film freezing (TFF). Previous studies reported that inhaled TAC combined with 50% w/w lactose (LAC) was safe and effective for the treatment of lung transplant rejection in rodent models. In this study, we aimed to investigate the safety and tolerability of TFF TAC-LAC in human subjects. The formulation can be delivered to the lung as colloidal dispersions after reconstitution and as a dry powder. Healthy subjects inhaled TAC-LAC colloidal dispersions at 3 mg TAC/dose via a vibrating mesh nebulizer in the first stage of this study and TAC-LAC dry powder at 3 mg TAC/dose via a single dose dry powder inhaler in the second stage. Our results demonstrated that oral inhalation of TAC-LAC colloidal dispersions and dry powder exhibited low systemic absorption. Additionally, they were well-tolerated with no changes in CBC, liver, kidney, and lung functions. Only mild adverse side effects (e.g., cough, throat irritation, distaste) were observed. In summary, pulmonary delivery of TFF TAC-LAC would be a safe and promising therapy for lung transplant recipients.

In COPD, prophylactic macrolides, but not tetracyclines or quinolones, reduce exacerbations, with fewer serious adverse events.

SOURCE CITATION: Janjua S, Mathioudakis AG, Fortescue R, et al. Prophylactic antibiotics for adults with chronic obstructive pulmonary disease: a network meta-analysis. Cochrane Database Syst Rev. 2021;1:CD013198. 33448349.

Niclosamide inhalation powder made by thin-film freezing: Multi-dose tolerability and exposure in rats and pharmacokinetics in hamsters.

In this work, we have developed and tested a dry powder form of niclosamide made by thin-film freezing (TFF) and administered it by inhalation to rats and hamsters to gather data about its toxicology and pharmacokinetics. Niclosamide, a poorly water-soluble drug, is an interesting drug candidate because it was approved over 60 years ago for use as an anthelmintic medication, but recent studies demonstrated its potential as a broad-spectrum antiviral with pharmacological effect against SARS-CoV-2 infection. TFF was used to develop a niclosamide inhalation powder composition that exhibited acceptable aerosol performance with a fine particle fraction (FPF) of 86.0% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 1.11 µm and 2.84, respectively. This formulation not only proved to be safe after an acute three-day, multi-dose tolerability and exposure study in rats as evidenced by histopathology analysis, and also was able to achieve lung concentrations above the required IC90 levels for at least 24 h after a single administration in a Syrian hamster model. To conclude, we successfully developed a niclosamide dry powder inhalation that overcomes niclosamide's limitation of poor oral bioavailability by targeting the drug directly to the primary site of infection, the lungs.

In spontaneous pneumothorax, initial needle aspiration and large- and narrow-bore chest tubes do not differ for success.

SOURCE CITATION: Mummadi SR, de Longpre' J, Hahn PY. Comparative effectiveness of interventions in initial management of spontaneous pneumothorax: a systematic review and a Bayesian network meta-analysis. Ann Emerg Med. 2020;76:88-102. 32115203.

Inhaled nanoparticles-An updated review.

We are providing an update to our previously published review paper on inhaled nanoparticles, thus updating with the most recent reports in the literature. The field of nanotechnology may hold the promise of significant improvements in the health and well-being of patients, as well as in manufacturing technologies. The knowledge of the impact of nanomaterials on public health is limited so far. This paper reviews the unique size-controlled properties of nanomaterials, their disposition in the body after inhalation, and the factors influencing the fate of inhaled nanomaterials. The physiology of the lungs makes it an ideal target organ for non-invasive local and systemic drug delivery, especially for protein and poorly water-soluble drugs that have low oral bioavailability via oral administration. More recently, inhaled nanoparticles have been reported to improve therapeutic efficacies and decrease undesirable side effects via pulmonary delivery. The potential application of pulmonary drug delivery of nanoparticles to the lungs, specifically in context of published results reported on nanomaterials in environmental epidemiology and toxicology is reviewed in this paper. This article presents updated delivery systems, process technologies, and potential of inhaled nanoparticles for local and systemic therapies administered to the lungs. The authors acknowledge the contributions of Wei Yang in our 2008 paper published in this journal.

COVID-19 in a Hispanic Woman.

Since making its debut on the global stage in December 2019, coronavirus disease 2019 (COVID-19) has afflicted nearly 4 million people and caused hundreds of thousands of deaths. Case reports and case series depicting the clinical effects of the causative virus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-have been published, yet few demonstrate the cytopathologic alterations of this disease. We present a clinical-pathologic correlation report of a previously healthy Hispanic woman with laboratory-confirmed COVID-19 who had typical features of acute respiratory distress syndrome (ARDS) and also showed cardiac abnormalities thought to represent fulminant viral myocarditis. Congruent with the ARDS clinical impression, autopsy findings were remarkable for extensive and markedly severe acute lung injury consistent with viral pneumonia, characterized by diffuse alveolar damage, pulmonary infarction, severe pulmonary edema, desquamation of pneumocytes with intra-alveolar aggregation, and pneumocyte morphologic alterations suggestive of viral cytopathic effect. However, there was incongruence between the clinical impression and the cardiovascular pathology findings in that viral myocarditis was not detected on histopathologic evaluation. This case highlights the importance of pathologic corroboration of the clinical impression and, in addition, illuminates the key role autopsy plays during a pandemic by providing valuable insight into viral pathology in tissues.

Successful management of fibrosing mediastinitis with severe vascular compromise: Report of two cases and literature review.

Fibrosing mediastinitis is a rare disorder characterized by the invasive proliferation of fibrous tissue within the mediastinum. This fibrosis can result in compression of intrathoracic structures including the pulmonary vasculature leading to clinical symptoms and complications like pulmonary hypertension. Here, we present two cases of young patients with fibrosing mediastinitis complicated by pulmonary artery stenosis requiring medical and endovascular management with excellent results.

Long-term evaluation of response to omalizumab therapy in real life by a novel multimodular approach: The Real-life Effectiveness of Omalizumab Therapy (REALITY) study.

BACKGROUND: The evidence on long-term real-life response measures to omalizumab therapy in moderate to severe asthma is limited. A universal assessment tool is needed to adequately evaluate response to omalizumab in these patients. OBJECTIVE: To design a multimodular response assessment tool and use it to measure and define response to omalizumab therapy in real-world settings. METHODS: The Real-life Effectiveness of Omalizumab Therapy (REALITY) study is a retrospective, long-term, real-life clinical study that evaluates response in individuals with allergic asthma who received omalizumab between 2004 and 2011. The Standardized Measure to Assess Response to Therapy (SMART) tool was designed to define response (1 year before to after treatment) by 3 modules: (1) physician's subjective assessment of asthma symptoms and control; (2) objective assessment of 6 parameters: improvement by 50% or more for asthma exacerbation, steroid bursts, emergency department visits, and hospitalizations; increase in forced expiratory volume in 1 second of 200 mL or greater; and improved Asthma Control Test score of 3 or higher; -and (3) true responders (patient meeting both module 1 and 2 criteria). Response was assessed and compared for 3 modules at desired time points. RESULTS: A total of 198 patients (mean age, 31.7 years [range, 3-77 years]; 98 [49%] female; mean omalizumab therapy duration, 2.49 years [range, 3 months to 8 years]; mean omalizumab dosage, 473 mg every 4 weeks; median baseline IgE level, 433 IU/mL) were included in this analysis. Overall visit adherence was 78%, although the adherence rate decreased annually by 20%. Response rates assessed by SMART modules were 61.3%, 60.8%, and 41.8% at 16 weeks, 84.8%, 72.2%, and 64.6% at 1 year, 82.4%, 71.2%, and 63.2% at 2 years, and 95.1%, 87.8%, and 85.4% at 5 years for modules 1, 2, and 3, respectively. There were no significant adverse reactions. CONCLUSION: The REALITY study has demonstrated long-term effectiveness of omalizumab therapy in individuals with allergic asthma in real-life settings. The SMART tool is promising as a potential standard assessment tool to measure and define response to asthma therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01776177.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.