The immunopathogenesis of sarcoidosis.

Sarcoidosis is a granulomatous multiorgan disease, thought to result from exposure to yet unidentified antigens in genetically susceptible individuals. The exaggerated inflammatory response that leads to granuloma formation is highly complex and involves the innate and adaptive immune system. Consecutive immunological studies using advanced technology have increased our understanding of aberrantly activated immune cells, mediators and pathways that influence the formation, maintenance and resolution of granulomas. Over the years, it has become increasingly clear that disease immunopathogenesis can only be understood if the clinical heterogeneity of sarcoidosis is taken into consideration, along with the distribution of immune cells in peripheral blood and involved organs. Most studies offer an immunological snapshot during disease course, while the cellular composition of both the circulation and tissue microenvironment may change over time. Despite these challenges, novel insights on the role of the immune system are continuously published, thus bringing the field forward. This review highlights current knowledge on the innate and adaptive immune responses involved in sarcoidosis pathogenesis, as well as the pathways involved in non-resolving disease and fibrosis development. Additionally, we describe proposed immunological mechanisms responsible for drug-induced sarcoid like reactions. Although many aspects of disease immunopathogenesis remain to be unraveled, the identification of crucial immune reactions in sarcoidosis may help identify new treatment targets. We therefore also discuss potential therapies and future strategies based on the latest immunological findings.

The future of clinical trials in idiopathic pulmonary fibrosis.

PURPOSE OF REVIEW: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and limited therapeutic options. A multitude of promising compounds are currently being investigated; however, the design and conductance of late-phase clinical trials in IPF has proven particularly challenging. RECENT FINDINGS: Despite promising phase 2 data, ziritaxestat, an autotaxin inhibitor, pentraxin-2, an endogenous protein that regulates wound healing and fibrosis, and pamrevlumab, a human monoclonal antibody against connective tissue growth factor, failed to show efficacy in phase 3 trials. Endpoint selection is critical for the design, execution, and success of clinical trials; recently, attention has been paid to the assessment of how patients feel, function, and survive with the aim of aligning scientific objectives and patient needs in IPF. External control arms are control patients that derive from historical randomized controlled trials, registries, or electronic health records. They are increasingly used to assess treatment efficacy in clinical trials owing to their potential to reduce study duration and cost and increase generalizability of findings. SUMMARY: Advances in study design, end point selection and statistical analysis, and innovative strategies for more efficient enrolment of study participants have the potential to increase the likelihood of success of late-phase clinical trials in IPF.

Occupational interstitial lung diseases.

Millions of workers are exposed to substances known to cause occupational interstitial lung diseases (ILDs), particularly in developing countries. However, the burden of the disease is likely to be underestimated due to under-recognition, under-reporting or both. The diagnosis of occupational ILD requires a high level of suspicion and a thorough occupational history, as occupational and non-occupational ILDs may be clinically, functionally and radiologically indistinguishable, leading to delayed diagnosis and inappropriate management. A potential occupational aetiology should always be considered in the differential diagnosis of ILD, as removal from the workplace exposure, with or without treatment, is a key therapeutic intervention and may lead to significant improvement. In this article, we provide an overview of the 'traditional' inorganic dust-related ILDs but also address idiopathic pulmonary fibrosis and the immunologically mediated chronic beryllium disease, sarcoidosis and hypersensitivity pneumonitis, with emphasis on the importance of surveillance and prevention for reducing the burden of these conditions. To this end, health-care professionals should be specifically trained about the importance of occupational exposures as a potential cause of ILD.

Walking the path of treatable traits in interstitial lung diseases.

Interstitial lung diseases (ILDs) are complex and heterogeneous diseases. The use of traditional diagnostic classification in ILD can lead to suboptimal management, which is worsened by not considering the molecular pathways, biological complexity, and disease phenotypes. The identification of specific "treatable traits" in ILDs, which are clinically relevant and modifiable disease characteristics, may improve patient's outcomes. Treatable traits in ILDs may be classified into four different domains (pulmonary, aetiological, comorbidities, and lifestyle), which will facilitate identification of related assessment tools, treatment options, and expected benefits. A multidisciplinary care team model is a potential way to implement a "treatable traits" strategy into clinical practice with the aim of improving patients' outcomes. Multidisciplinary models of care, international registries, and the use of artificial intelligence may facilitate the implementation of the "treatable traits" approach into clinical practice. Prospective studies are needed to test potential therapies for a variety of treatable traits to further advance care of patients with ILD.

Recent advances in the genetics of idiopathic pulmonary fibrosis.

PURPOSE OF REVIEW: Genetics contributes substantially to the susceptibility to idiopathic pulmonary fibrosis (IPF). Genetic studies in sporadic and familial disease have identified several IPF-associated variants, mainly in telomere-related and surfactant protein genes.Here, we review the most recent literature on genetics of IPF and discuss how it may contribute to disease pathogenesis. RECENT FINDINGS: Recent studies implicate genes involved in telomere maintenance, host defence, cell growth, mammalian target of rapamycin signalling, cell-cell adhesion, regulation of TGF-ß signalling and spindle assembly as biological processes involved in the pathogenesis of IPF. Both common and rare genetic variants contribute to the overall risk of IPF; however, while common variants (i.e. polymorphisms) account for most of the heritability of sporadic disease, rare variants (i.e. mutations), mainly in telomere-related genes, are the main contributors to the heritability of familial disease. Genetic factors are likely to also influence disease behaviour and prognosis. Finally, recent data suggest that IPF shares genetic associations - and probably some pathogenetic mechanisms - with other fibrotic lung diseases. SUMMARY: Common and rare genetic variants are associated with susceptibility and prognosis of IPF. However, many of the reported variants fall in noncoding regions of the genome and their relevance to disease pathobiology remains to be elucidated.

Lung cancer in patients with idiopathic pulmonary fibrosis: A retrospective multicentre study in Europe.

BACKGROUND AND OBJECTIVE: There remains a paucity of large databases for patients with idiopathic pulmonary fibrosis (IPF) and lung cancer. We aimed to create a European registry. METHODS: This was a multicentre, retrospective study across seven European countries between 1 January 2010 and 18 May 2021. RESULTS: We identified 324 patients with lung cancer among 3178 patients with IPF (prevalence = 10.2%). By the end of the 10 year-period following IPF diagnosis, 26.6% of alive patients with IPF had been diagnosed with lung cancer. Patients with IPF and lung cancer experienced increased risk of all-cause mortality than IPF patients without lung cancer (HR: 1.51, [95% CI: 1.22-1.86], p < 0.0001). All-cause mortality was significantly lower for patients with IPF and lung cancer with a monocyte count of either <0.60 or 0.60-<0.95 K/µl than patients with monocyte count ≥0.95 K/µl (HR [<0.60 vs. ≥0.95 K/µl]: 0.35, [95% CI: 0.17-0.72], HR [0.60-<0.95 vs. ≥0.95 K/µl]: 0.42, [95% CI: 0.21-0.82], p = 0.003). Patients with IPF and lung cancer that received antifibrotics presented with decreased all cause-mortality compared to those who did not receive antifibrotics (HR: 0.61, [95% CI: 0.42-0.87], p = 0.006). In the adjusted model, a significantly lower proportion of surgically treated patients with IPF and otherwise technically operable lung cancer experienced all-cause mortality compared to non-surgically treated patients (HR: 0.30 [95% CI: 0.11-0.86], p = 0.02). CONCLUSION: Lung cancer exerts a dramatic impact on patients with IPF. A consensus statement for the management of patients with IPF and lung cancer is sorely needed.

Integrating Clinical Probability into the Diagnostic Approach to Idiopathic Pulmonary Fibrosis: An International Working Group Perspective.

Background: When considering the diagnosis of idiopathic pulmonary fibrosis (IPF), experienced clinicians integrate clinical features that help to differentiate IPF from other fibrosing interstitial lung diseases, thus generating a "pre-test" probability of IPF. The aim of this international working group perspective was to summarize these features using a tabulated approach similar to chest HRCT and histopathologic patterns reported in the international guidelines for the diagnosis of IPF, and to help formally incorporate these clinical likelihoods into diagnostic reasoning to facilitate the diagnosis of IPF. Methods: The committee group identified factors that influence the clinical likelihood of a diagnosis of IPF, which was categorized as a pre-test clinical probability of IPF into "high" (70-100%), "intermediate" (30-70%), or "low" (0-30%). After integration of radiological and histopathological features, the post-test probability of diagnosis was categorized into "definite" (90-100%), "high confidence" (70-89%), "low confidence" (51-69%), or "low" (0-50%) probability of IPF. Findings: A conceptual Bayesian framework was created, integrating the clinical likelihood of IPF ("pre-test probability of IPF") with the HRCT pattern, the histopathology pattern when available, and/or the pattern of observed disease behavior, into a "post-test probability of IPF." The diagnostic probability of IPF was expressed using an adapted diagnostic ontology for fibrotic interstitial lung diseases. Interpretation: The present approach will help incorporate the clinical judgment into the diagnosis of IPF, thus facilitating the application of IPF diagnostic guidelines and, ultimately improving diagnostic confidence and reducing the need for invasive diagnostic techniques.

Environmental Causes of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF), the most common and severe of the idiopathic interstitial pneumonias, is a chronic and relentlessly progressive disease, which occurs mostly in middle-aged and elderly males. Although IPF is by definition "idiopathic", multiple factors have been reported to increase disease risk, aging being the most prominent one. Several occupational and environmental exposures, including metal dust, wood dust and air pollution, as well as various lifestyle variables, including smoking and diet, have also been associated with an increased risk of IPF, probably through interaction with genetic factors. Many of the predisposing factors appear to act also as trigger for acute exacerbations of the disease, which herald a poor prognosis. The more recent literature on inhalation injuries has focused on the first responders in the World Trade Center attacks and military exposure. In this review, we present an overview of the environmental and occupational causes of IPF and its pathogenesis. While our list is not comprehensive, we have selected specific exposures to highlight based on their overall disease burden.

Molecular Mechanism in the Development of Pulmonary Fibrosis in Patients with Sarcoidosis.

Sarcoidosis is a multisystemic disease of unknown etiology characterized by the formation of granulomas in various organs, especially lung and mediastinal hilar lymph nodes. The clinical course and manifestations are unpredictable: spontaneous remission can occur in approximately two thirds of patients; up to 20% of patients have chronic course of the lung disease (called advanced pulmonary sarcoidosis, APS) resulting in progressive loss of lung function, sometimes life-threatening that can lead to respiratory failure and death. The immunopathology mechanism leading from granuloma formation to the fibrosis in APS still remains elusive. Recent studies have provided new insights into the genetic factors and immune components involved in the clinical manifestation of the disease. In this review we aim to summarize the clinical-prognostic characteristics and molecular pathways which are believed to be associated with the development of APS.

Drug-induced interstitial lung disease.

Interstitial lung disease (ILD) secondary to drug-induced lung injury is an increasingly common cause of morbidity and mortality. The number of drugs associated with the development of ILD continues to rise, mainly due to the use of novel monoclonal antibodies and biologicals for neoplastic and rheumatological diseases, and includes, among others, chemotherapeutics, molecular targeting agents, immune checkpoint inhibitors, antibiotics, antiarrhythmics and conventional or biological disease-modifying antirheumatic drugs. Drug-induced ILD (DI-ILD) manifests with a variety of clinical patterns, ranging from mild respiratory symptoms to rapidly progressive respiratory failure and death. In most cases, there are no pathognomonic clinical, laboratory, radiological or pathological features and the diagnosis of DI-ILD is suspected in the presence of exposure to a drug known to cause lung toxicity and after exclusion of alternative causes of ILD. Early identification and permanent discontinuation of the culprit drug are the cornerstones of treatment with systemic glucocorticoids being used in patients with disabling or progressive disease. However, for certain drugs, such as checkpoint inhibitors, the frequency of lung toxicity is such that mitigation strategies are put in place to prevent this complication, and occurrence of DI-ILD is not necessarily synonymous with permanent drug discontinuation, particularly in the absence of valid therapeutic alternatives.

Revealing the pathogenic and ageing-related mechanisms of the enigmatic idiopathic pulmonary fibrosis (and chronic obstructive pulmonary disease).

PURPOSE OF REVIEW: Growing evidence suggests that ageing-associated alterations occur in both idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Here, we review the most recent literature on dysregulated ageing pathways in IPF and COPD and discuss how they may contribute to disease pathogenesis. RECENT FINDINGS: Recent studies have shown that alveolar epithelial type II (ATII) cells undergo premature senescence under stress and that senescent ATII cells promote lung fibrogenesis. Some studies have explored the role of mitochondrial dysfunction in IPF. They have provided evidence that dysfunctional mitochondria are important contributors to fibrogenesis through release of damaged DNA and excessive formation of reactive oxygen species, whereas restoration of mitochondrial homeostasis may attenuate lung fibrosis. Insufficient autophagy has been shown to promote epithelial-to-mesenchymal transition and aberrant epithelial-fibroblast crosstalk, suggesting that autophagy augmentation may represent a potential therapeutic strategy. A number of studies have also explored the role of cellular senescence, mitochondrial homeostasis and autophagy in COPD. SUMMARY: Several ageing mechanisms are dysregulated in the lungs of patients with IPF and COPD, although how they contribute to disease development and progression remains elusive. Genetic or pharmacologic attenuation of senescence-related pathways and elimination of senescent cells may represent a promising therapeutic strategy.

Pulmonary adverse events following immune checkpoint inhibitors.

PURPOSE OF REVIEW: Immune checkpoint inhibitors (ICIs) have rapidly become a mainstay of cancer treatment. However, immune modulation resulting from checkpoint inhibition can cause inflammation in any organ system, with pneumonitis being one of the most severe immune-related adverse events (irAEs). Here, we review the most recent literature on pulmonary adverse events following ICIs. RECENT FINDINGS: Several systematic reviews and meta-analyses of data from trials of antiprogrammed death-1 (PD-1; nivolumab, pembrolizumab), anti-PD-ligand-1 (PD-L1; atezolizumab, avelumab, durvalumab) and anticytotoxic T lymphocyte antigen-4 (CTLA-4; ipilimumab or tremelimumab) in patients with advanced cancer have explored the relative risk and incidence of lung toxicity among different tumor types and therapeutic regimens. They have showed that the incidence of all-grade (1-4) and high-grade (3-4) pneumonitis is significantly higher in nonsmall cell lung cancer (NSCLC) compared with other tumor types. In addition, they have demonstrated that immunotherapy, especially monoimmunotherapy, has a significantly lower risk of irAEs compared to immune-chemotherapy. Treatment for lung cancer, preexisting interstitial lung disease, smoking history and male sex appear to increase the risk for ICI-related pneumonitis. SUMMARY: Lung toxicity is an uncommon but potentially severe and even fatal complication of ICIs. Timely recognition is critically important but challenging, particularly in patients with lung cancer wherein drug toxicity can mimic disease progression or recurrence.

Radiological Assessment in Idiopathic Pulmonary Fibrosis (IPF) Patients According to MUC5B Polymorphism.

The MUC5B rs35705950 mutant T allele is the strongest genetic risk factor for familial and sporadic IPF. We sought to determine whether MUC5B genotype influences radiological patterns of IPF at diagnosis, as well as their change over time, in patients on antifibrotic therapy. Among eighty-eight IPF patients, previously genotyped for MUC5B rs35705950, we considered seventy-eight patients who were evaluated for radiological quantification of the following features both at treatment initiation (HRCT1) and after 1 year (HRCT2): ground glass opacities (AS), reticulations (IS) and honeycombing (HC). Of the evaluated patients, 69% carried at least one copy of the T allele (TT/TG). Carriers of the T allele displayed similar FVC loss in the first year of treatment as GG carriers, but overall survival at the end of follow-up was longer in the TT/TG group, compared to the GG group. In the GG group, both the AS and HC increased significantly, whereas in the TT/TG group only HC increased over the first year of treatment. MUC5B rs35705950 GG carriers are associated with increased ground glass and honeycombing extent over time and worse survival than T allele carriers. Longitudinal HRCT may help define the prognostic role of the MUC5B rs35705950 genotype.

Lung Microbiome in Idiopathic Pulmonary Fibrosis and Other Interstitial Lung Diseases.

Interstitial lung diseases represent a heterogeneous and wide group of diseases in which factors leading to disease initiation and progression are not fully understood. Recent evidence suggests that the lung microbiome might influence the pathogenesis and progression of interstitial lung diseases. In recent years, the utilization of culture-independent methodologies has allowed the identification of complex and dynamic communities of microbes, in patients with interstitial lung diseases. However, the potential mechanisms by which these changes may drive disease pathogenesis and progression are largely unknown. The aim of this review is to discuss the role of the altered lung microbiome in several interstitial lung diseases. Untangling the host-microbiome interaction in the lung and airway of interstitial lung disease patients is a research priority. Thus, lung dysbiosis is a potentially treatable trait across several interstitial lung diseases, and its proper characterization and treatment might be crucial to change the natural history of these diseases and improve outcomes.

RNA Sequencing of Epithelial Cell/Fibroblastic Foci Sandwich in Idiopathic Pulmonary Fibrosis: New Insights on the Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by irreversible scarring of the distal lung. IPF is best described by its histopathological pattern of usual interstitial pneumonia (UIP), characterized by spatial heterogeneity with alternating interstitial fibrosis and areas of normal lung, and temporal heterogeneity of fibrosis characterized by scattered fibroblastic foci (FF), dense acellular collagen and honeycomb changes. FF, comprising aggregated fibroblasts/myofibroblasts surrounded by metaplastic epithelial cells (EC), are the cardinal pathological lesion and their presence strongly correlates with disease progression and mortality. We hypothesized that the EC/FF sandwich from patients with UIP/IPF has a distinct molecular signature which could offer new insights into the crosstalk of these two crucial actors in the disease. Laser capture microdissection with RNAseq was used to investigate the transcriptome of the EC/FF sandwich from IPF patients versus controls (primary spontaneous pneumothorax). Differentially expressed gene analysis identified 23 up-regulated genes mainly related to epithelial dysfunction. Gene ontology analysis highlighted the activation of different pathways, mainly related to EC, immune response and programmed cell death. This study provides novel insights into the IPF pathogenetic pathways and suggests that targeting some of these up-regulated pathways (particularly those related to secreto-protein/mucin dysfunction) may be beneficial in IPF. Further studies in a larger number of lung samples, ideally from patients with early and advanced disease, are needed to validate these findings.

Chest radiography or computed tomography for COVID-19 pneumonia? Comparative study in a simulated triage setting.

INTRODUCTION: For the management of patients referred to respiratory triage during the early stages of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic, either chest radiography or computed tomography (CT) were used as first-line diagnostic tools. The aim of this study was to compare the impact on the triage, diagnosis and prognosis of patients with suspected COVID-19 when clinical decisions are derived from reconstructed chest radiography or from CT. METHODS: We reconstructed chest radiographs from high-resolution CT (HRCT) scans. Five clinical observers independently reviewed clinical charts of 300 subjects with suspected COVID-19 pneumonia, integrated with either a reconstructed chest radiography or HRCT report in two consecutive blinded and randomised sessions: clinical decisions were recorded for each session. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prognostic value were compared between reconstructed chest radiography and HRCT. The best radiological integration was also examined to develop an optimised respiratory triage algorithm. RESULTS: Interobserver agreement was fair (Kendall's W=0.365, p<0.001) by the reconstructed chest radiography-based protocol and good (Kendall's W=0.654, p<0.001) by the CT-based protocol. NPV assisted by reconstructed chest radiography (31.4%) was lower than that of HRCT (77.9%). In case of indeterminate or typical radiological appearance for COVID-19 pneumonia, extent of disease on reconstructed chest radiography or HRCT were the only two imaging variables that were similarly linked to mortality by adjusted multivariable models CONCLUSIONS: The present findings suggest that clinical triage is safely assisted by chest radiography. An integrated algorithm using first-line chest radiography and contingent use of HRCT can help optimise management and prognostication of COVID-19.

Mechanisms of progressive fibrosis in connective tissue disease (CTD)-associated interstitial lung diseases (ILDs).

Interstitial lung diseases (ILDs), which can arise from a broad spectrum of distinct aetiologies, can manifest as a pulmonary complication of an underlying autoimmune and connective tissue disease (CTD-ILD), such as rheumatoid arthritis-ILD and systemic sclerosis (SSc-ILD). Patients with clinically distinct ILDs, whether CTD-related or not, can exhibit a pattern of common clinical disease behaviour (declining lung function, worsening respiratory symptoms and higher mortality), attributable to progressive fibrosis in the lungs. In recent years, the tyrosine kinase inhibitor nintedanib has demonstrated efficacy and safety in idiopathic pulmonary fibrosis (IPF), SSc-ILD and a broad range of other fibrosing ILDs with a progressive phenotype, including those associated with CTDs. Data from phase II studies also suggest that pirfenidone, which has a different-yet largely unknown-mechanism of action, may also have activity in other fibrosing ILDs with a progressive phenotype, in addition to its known efficacy in IPF. Collectively, these studies add weight to the hypothesis that, irrespective of the original clinical diagnosis of ILD, a progressive fibrosing phenotype may arise from common, underlying pathophysiological mechanisms of fibrosis involving pathways associated with the targets of nintedanib and, potentially, pirfenidone. However, despite the early proof of concept provided by these clinical studies, very little is known about the mechanistic commonalities and differences between ILDs with a progressive phenotype. In this review, we explore the biological and genetic mechanisms that drive fibrosis, and identify the missing evidence needed to provide the rationale for further studies that use the progressive phenotype as a target population.

Prognostic role of MUC5B rs35705950 genotype in patients with idiopathic pulmonary fibrosis (IPF) on antifibrotic treatment.

BACKGROUND: A common variant located in the promoter region of MUC5B (rs35705950) is the strongest risk factor for sporadic and familiar IPF, as well as a predictor of outcome. However, there are no data on the effect of MUC5B rs35705950 genotype on the prognosis of IPF patients on antifibrotic treatment. The aim of this study is to determine, in a phenotypically well-characterized population of patients with IPF treated with antifibrotics, the impact of MUC5B rs35705950 genotype on disease progression and survival. METHODS: 88 IPF patients on antifibrotic treatment were followed-up from 2014 until transplantation, death or end of follow-up (December 2019). Disease progression was defined as a forced vital capacity (FVC) loss ≥ 5% per year. All patients were genotyped for MUC5B rs35705950 by PCR amplification and Sanger sequencing. RESULTS: Out of 88 patients, 61 (69%) carried the mutant T allele (TT or TG) and 27 (31%) did not (GG). Carriage of the MUC5B rs35705950 T allele was not associated with a faster decline in FVC. Conversely, at the end of the follow-up, overall survival in carriers of the TT/TG genotype was longer compared to that of the GG genotype carriers. FVC (L) at baseline and time to respiratory failure at rest were independent predictors of worse prognosis. CONCLUSIONS: In IPF patients on antifibrotic treatment, carriage of the MUC5B rs35705950 T allele is associated with longer survival, highlighting the usefulness of MUC5B genetic data in clinical decision making.

Genetics in sarcoidosis.

PURPOSE OF REVIEW: Epidemiological and clinical observations as well as familial clustering support the existence of a genetic predisposition to sarcoidosis. In this article, we review the most recent findings in genetics of sarcoidosis and discuss how the identification of risk alleles may help advancing our understanding of disease etiology and development. RECENT FINDINGS: Genetic studies of sarcoidosis phenotypes have identified novel and ancestry-specific associations. Gene-environment interaction studies highlighted the importance of integrating genetic information when assessing the relationship between sarcoidosis and environmental exposures. A case-control-family study revealed that the heritability of sarcoidosis is only 49%, suggesting the existence of additional important contributors to disease risk. The application of whole-exome sequencing has identified associations with disease activity and prognosis. Finally, gene expression studies of circulating immune cells have identified shared and unique pathways between sarcoidosis and other granulomatous diseases. SUMMARY: Sarcoidosis genetic research has led to the identification of a number of associations with both sarcoidoses per se and disease phenotypes. Newer sequencing technologies are likely to increase the number of genetic variants associated with sarcoidosis. However, studying phenotypically and ethnically homogeneous patient subsets remains critically important regardless of the genetic approach used.

Critical Review of the Evolution of Extracellular Vesicles' Knowledge: From 1946 to Today.

Extracellular vesicles (EVs) are a family of particles/vesicles present in blood and body fluids, composed of phospholipid bilayers that carry a variety of molecules that can mediate cell communication, modulating crucial cell processes such as homeostasis, induction/dampening of inflammation, and promotion of repair. Their existence, initially suspected in 1946 and confirmed in 1967, spurred a sharp increase in the number of scientific publications. Paradoxically, the increasing interest for EV content and function progressively reduced the relevance for a precise nomenclature in classifying EVs, therefore leading to a confusing scientific production. The aim of this review was to analyze the evolution of the progress in the knowledge and definition of EVs over the years, with an overview of the methodologies used for the identification of the vesicles, their cell of origin, and the detection of their cargo. The MISEV 2018 guidelines for the proper recognition nomenclature and ways to study EVs are summarized. The review finishes with a "more questions than answers" chapter, in which some of the problems we still face to fully understand the EV function and potential as a diagnostic and therapeutic tool are analyzed.