A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a crossdisciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoption of MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data. ' 2008 International Society for Advancement of Cytometry Key termsimmunology; fluorescence-activated cell sorting; knowledge representation FLOW cytometry (FCM) systems have been available to investigators for over 30 years, and the field continues to advance at a rapid rate. FCM has been responsible for major progress in basic and clinical research by enabling the phenotypic and functional characterization of individual cells in a high-throughput manner. Advances in the technology now allow for automated, multiparametric analyses of thousands of samples per day (1). Each data set can consist of multidimensional descriptions of millions of individual cells, producing data similar in size and complexity to gene expression microarrays. Like the microarray field, the ability to collect FCM data is outpacing the computational means for data handling and analysis. Furthermore, the lack of reporting standardization limits collaboration, independent validation/refutation, and meta-analysis, and thus minimizes the value of the wealth

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Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.

Oral tolerance can develop after frequent exposure to food allergens. Upon ingestion, food is digested into small protein fragments in the gastrointestinal tract. Small food particles are later absorbed into the human body. Interestingly, some of these ingested food proteins can cause allergic immune responses, which can lead to food allergy. So far it has not been completely elucidated how these proteins become immunogenic and cause food allergies. In contrast, oral tolerance helps to prevent the pathologic reactions against different types of food antigens from animal or plant origin. Tolerance to food is mainly acquired by dendritic cells, epithelial cells in the gut, and the gut microbiome. A subset of CD103+ DCs is capable of inducing T regulatory cells (Treg cells) that express anti-inflammatory cytokines. Anergic T cells also contribute to oral tolerance, by reducing the number of effector cells. Similar to Treg cells, B regulatory cells (Breg cells) suppress effector T cells and contribute to the immune tolerance to food allergens. Furthermore, the human microbiome is an essential mediator in the induction of oral tolerance or food allergy. In this review, we outline the current understanding of regulatory immune mechanisms in oral tolerance. The biological changes reflecting early consequences of immune stimulation with food allergens should provide useful information for the development of novel therapeutic treatments.

B cells play a central role in the immune system through the production of antibodies. During the past two decades, it has become increasingly clear that B cells also have the capacity to regulate immune responses through mechanisms that extend beyond antibody production. Several types of human and murine regulatory B cells have been reported that suppress inflammatory responses in autoimmune disease, allergy, infection, transplantation, and cancer. Key suppressive molecules associated with regulatory B-cell function include the cytokines IL-10, IL-35, and TGFβ as well as cell membrane-bound molecules such as programmed death-ligand 1, CD39, CD73, and aryl hydrocarbon receptor. Regulatory B cells can be induced by a range of different stimuli, including microbial products such as TLR4 or TLR9 ligands, inflammatory cytokines such as IL-6, IL-1β, and IFNα, as well as CD40 ligation. This review provides an overview of our current knowledge on regulatory B cells. We discuss different types of regulatory B cells, the mechanisms through which they exert their regulatory functions, factors that lead to induction of regulatory B cells and their role in the alteration of inflammatory responses in different diseases.

In this review, we discuss recent publications on asthma and review the studies that have reported on the different aspects of the prevalence, risk factors and prevention, mechanisms, diagnosis, and treatment of asthma. Many risk and protective factors and molecular mechanisms are involved in the development of asthma. Emerging concepts and challenges in implementing the exposome paradigm and its application in allergic diseases and asthma are reviewed, including genetic and epigenetic factors, microbial dysbiosis, and environmental exposure, particularly to indoor and outdoor substances. The most relevant experimental studies further advancing the understanding of molecular and immune mechanisms with potential new targets for the development of therapeutics are discussed. A reliable diagnosis of asthma, disease endotyping, and monitoring its severity are of great importance in the management of asthma. Correct evaluation and management of asthma comorbidity/ multimorbidity, including interaction with asthma phenotypes and its value for the precision medicine approach and validation of predictive biomarkers, are further detailed. Novel approaches and strategies in asthma treatment linked to mechanisms and endotypes of asthma, particularly biologicals, are critically appraised. Finally, due to the recent pandemics and its impact on patient management, we discuss the challenges, relationships, and molecular mechanisms between asthma, allergies, SARS-CoV-2, and COVID-19.

and by the ''Angelo Nocivelli'' Foundation. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

In this review, we discuss recent publications on asthma and review the studies that have reported on the different aspects of the prevalence, risk factors and prevention, mechanisms, diagnosis and treatment of asthma. Many risk and protective factors and molecular mechanisms are involved in the development of asthma. Emerging concepts and challenges in implementing the exposome paradigm and its application in allergic diseases and asthma are reviewed, including genetic and epigenetic factors, microbial dysbiosis and environmental exposure, particularly to indoor and outdoor substances. The most relevant experimental studies further advancing the understanding of molecular and immune mechanisms with potential new targets for the development of therapeutics are discussed. A reliable diagnosis of asthma, disease endotyping and monitoring its severity are of great importance in the management of asthma. Correct evaluation and management of asthma comorbidity/multimorbidity, including interaction with asthma phenotypes and its value for the precision medicine approach and validation of predictive biomarkers are further detailed. Novel approaches and strategies in asthma treatment linked to mechanisms and endotypes of asthma, particularly biologicals, are critically appraised. Finally, due to the recent pandemics and its impact on patient management, we discuss the challenges, relationships, and molecular mechanisms between asthma, allergies, SARS-CoV-2 and Covid-19.