The gut microbiota is often mentioned as a “forgotten organ” or “metabolic organ”, given its profound impact on host physiology, metabolism, immune function and nutrition. A healthy diet is undoubtedly a major contributor for promoting a “good” microbial community that turns out to be crucial for a fine-tuned symbiotic relationship with the host. Both microbial-derived components and produced metabolites elicit the activation of downstream cascades capable to modulate both local and systemic immune responses. A balance between host and gut microbiota is crucial to keep a healthy intestinal barrier and an optimal immune homeostasis, thus contributing to prevent disease occurrence. How dietary habits can impact gut microbiota and, ultimately, host immunity in health and disease has been the subject of intense study, especially with regard to metabolic diseases. Only recently, these links have started to be explored in relation to lung diseases. The objective of this review is to address the current knowledge on how diet affects gut microbiota and how it acts on lung function. As the immune system seems to be the key player in the cross-talk between diet, gut microbiota and the lungs, involved immune interactions are discussed. There are key nutrients that, when present in our diet, help in gut homeostasis and lead to a healthier lifestyle, even ameliorating chronic diseases. Thus, with this review we hope to incite the scientific community interest to use diet as a valuable non-pharmacological addition to lung diseases management. First, we talk about the intestinal microbiota and interactions through the intestinal barrier for a better understanding of the following sections, which are the main focus of this article: the way diet impacts the intestinal microbiota and the immune interactions of the gut–lung axis that can explain the impact of diet, a key modifiable factor influencing the gut microbiota in several lung diseases. Thethe host .In recent decades, multiomics techniques  became advanced and powerful tools capable of exploring the complex gut microbial ecosystem, namely its structure and function , such as lipopolysaccharides (LPS), peptidoglycans or flagellin, through pattern recognition receptors (PRRs) . PRRs in+ T cells that secrete a wide range of cytokines ), with a vital role in host defence against various types of infectious pathogens and involved in different types of tissue injury in immunologic diseases [The commensal microbiota plays a central role by reinforcing barrier immunity while protecting their own ecological niche against opportunistic pathogens ,33. For diseases ,40,41,42diseases ,27. As adiseases .Even though there is a vast density of microbial cells in the gut, it is unusual for commensal bacteria to breach the intestinal barrier, therefore avoiding tissue inflammation and microbial translocation and maintaining the mutualistic relationship between host and the gut microbiota. The homeostasis is preserved by several layers of defence against microbial translocation. In the first layer, termed “mucosal firewall”, the release of immune mediators restricts the direct contact of commensals to the epithelial cell surface and penetration, avoiding further immune activation and inflammation. The second layer of immune protection comprises the detection and killing of bacteria that penetrate the epithelium. Finally, the third layer, named “immune firewall”, reduces the exposure of commensal microbes to the systemic immune system, by inducing adaptive immune responses with the purpose to confine the bacteria in the luminal or mucosal compartments ,32,44,45Salmonella spp.) can evade phagocytic killing by downregulating biocidal mechanisms of macrophages [These layers include several structural and immunological constituents . Besidesrophages ,32. Finarophages ,44. The loss of the intestinal barrier integrity facilitates translocation of deleterious components  which are important players in pathogenesis of many diseases, thereby associating with various intestinal diseases, such as IBD, and systemic and metabolic disorders, like obesity or diabetes. A wide range of factors can compromise intestinal homeostasis and increase the permeability of the barrier that includes the presence of pathogens, environmental stress, high-fat/high-sugar diets, drugs and antibiotics, among others .Diet has been considered a major environmental factor affecting gut microbiota composition. Pinpointing the exact interactions between dietary components and microbiota might offer clues towards a better understanding of disease pathogenesis, enabling improved strategies for prevention and treatment . The impRecent findings are beginning to untangle a link between gut microbiota and lung immunity, the gut–lung axis ,52,53. IThe dynamics and intimate relationship occurring in the lung-gut axis could be partially derived from the fact that both gastrointestinal and respiratory mucosal tracts share the same origin and aspects of physiology and structure, such as the direct contact to the mouth and pharynx. Both have a physical barrier with projections of microvilli (gut) or cilia (respiratory tract) that also participate in conjunction with lymphoid tissues to the local immune system. Besides this, they also share secretory IgA and mucus-producing goblet cells. Indeed, they derive from the same common embryonic organ, the foregut ,57,58.The cross-talk between the gut and the lungs occurs through lymph and bloodstream circulatory systems and is crucial for passing long-reaching “immunological information” between organs ,59,60. TSCFAs, mainly propionate, acetate and butyrate, resulting from the fermentation of undigested soluble dietary fibres by the gut microbiota, are important immunomodulatory metabolites and their role has been extensively explored in the gut–lung axis. SCFAs exert direct or indirect effects on the function of several cells including epithelial, innate and adaptive immune cells .SCFAs mostly exert anti-inflammatory effects in the intestine by modulating epithelial and immune cell functions through the activation of G protein-coupled receptors 41, 43 and 109A  and the inhibition of histone deacetylases ,66. As eThe direct role of SCFAs in the lungs is most likely not substantial as circulating SCFAs do not seem to accumulate in the airways and there is no significant production of SCFAs in place. As such, it is feasible that SCFAs exert a direct effect on immune cells in the periphery with later recruitment to the lungs ,63,69. RClostridium orbiscindens during flavonoid metabolism, augments type I IFN signalling with a consequent reduction of lung immunopathology  during influenza virus infection [+ T cell polarisation into Th17 response in the lungs of mice, in response to pulmonary fungal infections (Aspergillus fumigatus). The increase of Th17 cell counts in the lungs by SFB provided antifungal immunity and was dependent on IL-1 receptor ligands, with authors suggesting that SFB may promote the systemic release of IL-1 receptor ligands [Candida albicans was found to drive potent CD4+ T-cell-mediated allergic airway responses to A. fumigatus, according to Noverr et al. [Other mechanisms of the long-reaching immune modulation include the intestinal microbial metabolite desaminotyrosine (DAT) and the commensal gut segmented filamentous bacteria (SFB) ,71. DAT,nfection . SFB appnfection ,73. For r et al. . Furtherr et al. . Escherichia coli pneumonia by TLR4 signalling in mice, namely, by increasing neutrophils infiltration and the killing activity of alveolar macrophages [Streptococcus pneumoniae and Staphylococcus aureus) [In addition, the migration of immune cells from the gut to the lungs as well as MAMPs seem to be other important players in the long-reaching immune interactions . The immrophages . Finally aureus) .The interactions discussed above sustain a central role for the gut–lung axis in lung health and disease.Changes in gut microbiota composition triggered by diet have been documented in chronic lung diseases and respiratory acute infections. Several studies are beginning to unravel the importance of a window of opportunity during which changes in the microbiota will have an impact on long-term health.Asthma is the most common noncommunicable disease among children. Worldwide, it affects more than 339 million people, being a significant public health burden . BecauseWhen one talks about diet and how it modulates the composition of the intestinal microbiota with inherent health consequences, namely, predisposition for allergy and asthma, we can point one of the first most striking moments in the early life of a human being: breastfeeding. Breastfeeding shapes the neonate´s gut microbiota in virtue of exposure to the milk microbiota but also to other components such as human milk oligosaccharides, secretory IgA and anti-microbial factors . BreastmLachnospira, Veillonella, Faecalibacterium and Rothia were significantly reduced in children at risk of asthma. Along with the reduction in bacterial taxa, reduction of faecal acetate and dysregulation of enterohepatic metabolites were observed [Bifidobacterium, Akkermansia and Faecalibacterium) and higher relative abundance of specific fungi (Candida and Rhodotorula) and a fecal metabolome enriched for pro-inflammatory metabolites [Lachnobacterium, Lachnospira and Dialister were lower abundant taxa throughout infancy among children who develop asthma [Akkermansia muciniphila and Faecalibacterium prausnitzii compared to healthy controls. Authors suggest that both bacterial species may induce anti-inflammatory cytokine IL-10 production and prevent the secretion of pro-inflammatory cytokine IL-12 through its secreted metabolites [Some studies highlight the importance of a window of opportunity, in the neonatal period, during which changes in the microbiota will be critical in long-term outcomes, such as predisposing subjects to develop atopy and asthma . In a loobserved . Similarabolites . More rep asthma . Furtherabolites .Faecalibacterium and Lachnospira, which inversely associate with asthma, are butyrate producers utilising the butyrogenic pathway (from pyruvate), which is a marker for gut commensals [Faecalibacterium has been shown to produce a 15 kDa protein with anti-inflammatory properties and capable not only to suppress the NF-κB pathway in intestinal epithelial cells and but also to prevent colitis in mice [Lachnospira and, to a lesser account, Lachnobacterium produce acetate. Acetate has been shown to associate with a marked reduction of allergic airway disease in a human asthma mouse-model by enhancing Treg cell numbers and function [Notably, both genera mmensals . Additio in mice . In addifunction . In a birth cohort involving children from rural areas of five European countries , SCFAs were measured in stools from 301 one-year-old children. Infants with high amounts of butyrate and propionate in faeces had significantly less atopic sensitisation and were less likely to develop asthma between 3 and 6 years of age . FurtherA more westernised diet  has been adopted in the past few decades coinciding with the increased prevalence of inflammatory and autoimmune diseases, including asthma ,96.Some studies have shed light on the role of fibre intake on promoting both a healthy gut microbiota composition and an enhanced production of SCFA, leading to amelioration of asthmatic symptoms. In line with this, other studies analysed the direct association between SCFA increment and reduction of airway inflammation in asthma.A pilot study conducted in Australia, involving 29 adult patients with asthma, found that 4 h after the intake of a 3.5 g dose of a soluble fibre, inulin, there was a significant reduction in airway inflammation, namely, by reduction in sputum total cell count, neutrophils, macrophages, lymphocytes, IL-8 and a marker for eosinophilic inflammation, exhaled nitric oxide. Interestingly, this was accompanied by upregulation of GPR41 and GPR43 sputum cell gene expression and an improvement in lung function. Authors further suggest that fermentation of inulin results in the activation of both receptors in immune cells in the airways . F. prausnitzii, when compared to healthy counterparts. This suggests that reduction of bacteria related to SCFA metabolism in asthma might contribute to the inflammatory setting in the pathogenesis of asthma [A cohort of 36 adult UK asthma-patients presented a decrease in both the gut microbial richness and the abundance of certain butyrate-producing bacteria, such as f asthma . Metabolism of a high-fat meal triggers innate immune responses via TLR activation with subsequent increase in pro-inflammatory cytokine production, as observed in an Australian study involving 37 non-obese adults with asthma and 14 obese patients with asthma. Four hours after consumption of a high-fat meal , patients with asthma had increased airway inflammation as seen by the increase in neutrophils and TLR4 mRNA expression in sputum, when compared to asthmatic patients given a low-fat meal. The high-fat meal suppressed bronchodilator recovery in asthma as inferred by the forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC) ratio. Moreover, when comparing effects from meals with high-trans or non-trans fatty acids, sputum neutrophils were significantly higher with high-trans fatty acids . It is fA recent analysis integrating dietary, microbiome and plasma metabolomics into an evaluation of the intestinal metabolome in 361 3-year old-children in the USA, revealed inverse associations between asthma and intestinal polyunsaturated fatty acids (PUFAs), which are likely representing dietary PUFAs both in excess of the amount absorbed in the small intestine and non-metabolised by the gut microbiota. Intestinal PUFAs inversely correlated with the asthma-associated intestinal microbiome. Furthermore, a diet rich in fried and processed meats was linked with asthma and with asthma-associated intestinal metabolites . Chronic obstructive pulmonary disease (COPD) is a progressive chronic lung disease estimated to affect 4–5% of the global population and to cause 2.9 million deaths annually. COPD is characterised by prolonged airway inflammation, lung tissue damage (emphysema) and airflow obstruction ,57. Haemophilus and Moraxella (Proteobacteria) were positively associated with disease severity and exacerbation events. These genera may be involved in biofilm formation and antibiotic resistance [A UK prospective, observational cohort study involving 101 COPD patients found that instability of the lung microbiota composition was linked with disease exacerbations. The bacterial genera sistance .COPD associates with multiple factors such as tobacco smoking and the use of antibiotics and oral corticosteroids, which are known to cause gut dysbiosis. Therefore, besides lung microbial dysbiosis, the occurrence of gut microbial dysbiosis in COPD patients is expected . IncreasStreptococcus species  were substantially higher in COPD patients, being identified as key differentiators between COPD and healthy groups. Several species, including Streptococcus sp000187445, Streptococcus vestibularis and various members of the family Lachnospiraceae, also associated with reduced lung function. Among the metabolites significantly diverging between COPD and healthy subjects, N-carbamoylglutamate, which has been associated with omega-3 fatty acid intake in humans, was reduced in COPD. Authors comment that further work is necessary to pinpoint whether the identified species are contributing to the airway neutrophilia and lung function decline in COPD [In a very recent Australian study, the faecal microbiome and metabolome were found to be significantly different between COPD patients (28 subjects) and healthy controls (29 subjects). A total of 146 bacterial species diverged between the two groups.  in COPD . A UK cohort study, with 1551 males and 1391 females, analysed the relationship between dietary patterns, lung function and COPD. The methodology included a complete spirometry and dietary data. A “prudent” dietary pattern  was correlated with protection against impaired lung function and COPD, particularly in male smokers . AuthorsThe high intake of dietary fibre has been recently suggested to become a therapeutic intervention in COPD as a mean to reduce chronic airway inflammation by promoting a healthy gut microbiota and increased SCFA production . Lately,Pseudomonas [Cystic Fibrosis (CF) is a progressive autosomal recessive disease characterised by a dysfunction in a chloride channel, the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR), resulting in thick and sticky mucus obstructing the airways, for instance. The lungs are among the multiple organs the most severely affected, with serious lung infections like udomonas . Eubacterium rectale and Faecalibacterium prausnitzii (both butyrate producers), Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium catenulatum were all reduced in children with CF. Intestinal inflammation, assessed by fecal calprotectin (CLP) and rectal nitric oxide (rNO) levels, was significantly higher in CF as compared to controls. Possibly, the depletion in CF of some of the butyrate producers presented above may explain the increase in inflammation due to lower butyrate levels. Interestingly, administration of the probiotic Lactobacillus GG partially restored the composition of intestinal microbiota, followed by a reduction in intestinal inflammation [An Italian randomised clinical trial, enrolling 22 children with CF compared with healthy controls, found a disrupted intestinal microbiota in CF. CF patients had reduced microbial diversity. At the species level, ammation . An Amerammation .Roseburia and a significant increase in Veillonella were detected in the cohort with CF. Additionally, in vitro data demonstrated that apical exposure of intestinal epithelial cells to secreted products from multiple beneficial Bacteroides species reduces IL-8 production, thus suggesting a mechanism by which the gut microbiota may influence systemic signals of inflammation in CF patients and potentially impact airway disease. Considering this, authors suggest the need for CF therapeutics that direct gastrointestinal microbiota towards a healthier state [Roseburia, Dorea, Sporacetigenium, Coprococcus, Blautia, Enterococcus and Escherichia) presaged their appearance in the respiratory tract and their abundances in both compartments were interrelated over time. Breast milk exposure had an impact on respiratory tract as indicated by the lung bacteria profiles related with breast-feeding. Overall, these findings highlight a potential link between nutrition and gut colonisation patterns as determinants of the lung microbial development in CF infants [Some studies disclose possible interactions of gut–lung axis within CF context . The stoer state . In anot infants . Recently, the nutrient intake and adherence to dietary recommendations were assessed in 76 CF Greek children and adolescent. Despite an optimal adherence to the energy and fat recommendations for this disease, most of the subjects had a low intake of carbohydrates and fibres and revealed a poor adherence to the Mediterranean diet . This imDiet is a readily modifiable factor influencing the gut microbiota. Research focusing on how the gut–lung axis acts on local and systemic immunity and the subsequent effects on human health is still in its infancy. Increasing recent evidence starts to unveil the impact of both nutrition and gut microbiota in the respiratory function. A diet rich in fibres, such as the Mediterranean diet, promotes a healthy gut microbiota composition, enhancing production of SCFAs, like butyrate. The beneficial impact on gut microbiota and its metabolites improves lung function in numerous diseases, such as asthma or COPD, by reducing local and systemic inflammation. The diet itself appears to modulate directly the lung microbiota as well. Importantly, diet is one of the most readily accessible interventions for everyone to prevent and/or ameliorate a wide range of diseases. For this fact and from the accumulating scientific evidence, we conclude that diet and nutrition should not be neglected when one talks about respiratory diseases. As such, we recommend diet as a fundamental non-pharmacological addition to lung diseases management. Gathering the already existing evidence and writing specific dietary guidelines for each respiratory disease would benefit patients and reduce the global burden of disease. Nutritional advice by clinical experts should be considered in cooperation with the already in place clinical consultations . Curiously, very recently, it has been suggested that gut microbiota dysbiosis could contribute to severity and persistency of symptoms of COVID-19 (primarily a respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)), possibly via modulating host immune responses . Indeed,Although a large number of studies have focused in recent years on the gut–lung axis and the role of dietary components, detailed mechanistic studies and clinical intervention trials with thoroughly assessed clinical outcomes are still needed to gain insight on the intricate associations going on between diet and gut and lung microbiota composition and metabolism and their influence on the immune system. Apart from metagenomics, the functional analyses by metabolomics and metatranscriptomics should be considered for accurately pinpointing the mechanisms connecting food intake to changes in human health.Lactobacillus rhamnosus GG has been reported to delay or reduce the occurrence of asthma in children [Besides diet, probiotic intake and other factors to manipulate the gut microbiota, microbial products or metabolites could be possibly used in the clinic to favour a healthy state. Probiotics may be cost-effective regarding prevention and treatment of some diseases; however, since they usually fall into the regulatory category of food or dietary supplements , they archildren ,123. Thechildren .Besides this, other components of the gut microbiota should be explored in future studies, specifically the mycobiota and virome, which are currently largely unexplored. Their role in the gut–lung axis should be examined with the aim of finding new therapeutic strategies to improve lung diseases. Finally, it must be stressed that the gut–lung axis is bidirectional and, although this review has focused on the impact of the intestine on the lung, the reverse has also been demonstrated. It is now well established that chronic and acute lung disease trigger changes in the gut microbiology and physiology . The lun


Cold physical plasma is a partially ionized gas generating various reactive oxygen and nitrogen species (ROS/RNS) simultaneously. ROS/RNS have therapeutic effects when applied to cells and tissues either directly from the plasma or via exposure to solutions that have been treated beforehand using plasma processes. This review addresses the challenges and opportunities of plasma-treated solutions (PTSs) for cancer treatment.Cold physical plasma is a partially ionized gas generating various reactive oxygen and nitrogen species (ROS/RNS) simultaneously. ROS/RNS have therapeutic effects when applied to cells and tissues either directly from the plasma or via exposure to solutions that have been treated beforehand using plasma processes. This review addresses the challenges and opportunities of plasma-treated solutions (PTSs) for cancer treatment. These PTSs include plasma-treated cell culture media in experimental research as well as clinically approved solutions such as saline and Ringer’s lactate, which, in principle, already qualify for testing in therapeutic settings. Several types of cancers were found to succumb to the toxic action of PTSs, suggesting a broad mechanism of action based on the tumor-toxic activity of ROS/RNS stored in these solutions. Moreover, it is indicated that the PTS has immuno-stimulatory properties. Two different routes of application are currently envisaged in the clinical setting. One is direct injection into the bulk tumor, and the other is lavage in patients suffering from peritoneal carcinomatosis adjuvant to standard chemotherapy. While many promising results have been achieved so far, several obstacles, such as the standardized generation of large volumes of sterile PTS, remain to be addressed. Cancer is a devastating condition, and the second leading cause of death in Western countries . DespiteCold physical plasma, which also goes by low-temperature plasma or nonthermal plasma, is a partially ionized gas generating a multitude of ROS/RNS simultaneously. More than two decades ago, it was proposed that these ROS/RNS can be used for therapeutic purposes. Early work focused on plasma-based disinfection of biologically contaminated surfaces and liquids ,6, whichWhile the direct application of cold physical plasma to cells and tissues is promising and approved in European dermatology centers , an incrSuch treated liquids go by several terms, such as plasma-activated medium (PAM) , plasma-It has been reported that PTSs selectively kill glioblastoma brain tumor cells against astrocyte normal cells . AntitumFor cell culture media, reaction products are complex since they contain about 30 components. However, several components such as amino acids  and fetaCold physical plasma interacts with oxygen, nitrogen, and water in the air and generates short-lived reactive species in the gas phase such as nitric oxide, ozone, hydroxyl radicals, singlet oxygen, and superoxide anion . Long-li2O2 ..74].2O2 generation in PTS, which further affects the anticancer performance . Th. Th86]. In a recent report, we used a PTS  to investigate its toxicity in four different gastrointestinal tumor cell lines in vitro. In addition to analyzing the liquid chemistry, we observed the onset of ICD in CT26 colorectal cancer cells in vitro. Strikingly, this was accompanied by a massive antitumor action of PTS  in vivo and an increase in intratumoral macrophages as well as T cells , which cAs concluded from above, the oxidative stress inflicted upon tumor cells using PTSs has an immuno-modulatory dimension. The extent of this dimension is underexplored at the moment. The main idea is that the PTS-induced tumor cell death is immunogenic, thereby promoting the immune system to recognize dying tumor cells in an inflammatory context with subsequent induction and promotion of antitumor immunity. The exact requirements for PTS to induce such an effect remain to be elucidated. However, from the in vivo studies mentioned above, it is clear that PTS exposure of gastrointestinal tumor burden increases influx of professional antigen-presenting cells such as dendritic cells and macrophages. It is also possible that the immune cells present within the TME will be directly by PTS. As mentioned above, T cells severely succumb to oxidation-induced toxicity. Data are intratumoral immune cells and their cell death after PTS exposure has not been reported so far.Another point is that the anticancer mechanisms of PTSs are of a general nature—it is uncertain as to whether they are more potent in some tumor entities over others. Along similar lines, great heterogeneity is observed within individual tumor entities, which is related to different mutational loads, variation in cell types within the TME, and signaling pathway deregulation. The current response rates to conventional drugs and immunotherapy show that responders and nonresponders can be stratified according to these factors, and it seems likely that PTS therapy will face similar drawbacks. However, progress of the PTS regimen to clinics is absent at the moment, leaving such aspects to be debated in the future. Comparative preclinical studies that incorporate different tumor entities or cell types with different traits of the same entity would greatly facilitate a much-needed gain in the knowledge in the field of PTSs. Such studies should also elaborate on the “dosing” of PTS—i.e., whether hormetic effects are observed that might show subtoxic effects at low doses.The safety of a PTS (water) in immune-deficient nude mice was investigated using this PTS by oral lavage treatment . The acuThe repeated application of PTSs  in the peritoneal cavity of tumor-bearing mice suffering from peritoneal pancreatic carcinomatosis was tolerated well without visible side effects. The relative frequencies of several blood parameters were not affected in the PTS group compared to the vehicle group. This was the case for neutrophils, monocytes, lymphocytes, T helper cells, cytotoxic T cells, B cells, and NK cells. For the non-nucleated blood cells (erythrocytes and thrombocytes), similar counts and volumes were determined in both groups. The quantification of six different chemokines and cytokines from nonstimulated and LPS-stimulated splenocytes of both groups also did not show any differences . Hence, Plasma-treated solutions (PTSs) show potent antitumor efficacy in many cancer cell lines in vitro and in vivo. Cutting-edge research currently addresses the immuno-modulating effects of the exposure of PTS to cancer cells and immune cells of the tumor microenvironment. A future step is to increase the translation of this promising field into clinics using solutions accredited for administration in humans. Along those lines, large bulk plasma-generators and ISO-standardized processes are needed to generate sufficient amounts of PTS in a reproducible and quality-controlled manner.


Drosophila melanogaster that illustrate non-apoptotic roles of apoptotic caspases. We will preface these examples with similar observations in other experimental systems and end with a discussion of how apoptotic caspase activity might be constrained to provide non-lethal functions without killing the cell.An increasing number of studies demonstrate that cells can activate apoptotic caspases but not die and, instead, display profound changes in cellular structure and function. In this minireview, we will discuss observations in the nervous system of  Drosophila melanogaster (Drosophila Death related ced-3/Nedd2-like caspase (Dredd), have dedicated roles in non-apoptotic processes such as inflammation and immunity. Other members, for example human caspases 3 and 9 and Drosophila Death regulator Nedd2-like caspase (Dronc) and Death related ICE-like caspase (Drice), are essential for apoptosis and will be referred to as “apoptotic caspases.” An increasing body of literature, however, documents non-apoptotic functions of apoptotic caspases, in altering cell identity, sub-cellular remodeling, and production of extra-cellular signals, to name a few. Such apoptotic caspase-driven alterations in cellular structure and function are found in different cell types and across diverse organisms. Here, we will use examples from the Drosophila nervous system to illustrate multiple non-apoptotic roles of apoptotic caspases. We will spring-board off excellent reviews on the subject . Caspant cells  and Abnoelopment . Likewisnumb mutant. This rescue required the presence of wild type Numb, suggesting that the rescue occurs through sequestration of mutant Numb to allow the wildtype Numb to work. The rescue did not require effector caspase activity or the catalytic activity of Dronc, suggesting that Dronc acts in a non-apoptotic manner. Full length Dronc binds Numb as well as cleaved active Dronc, suggesting that any activity provided by Dronc to limit neuroblast number can occur without fear of killing the cell. dronc loss-of-function mutants do not have extra neuroblasts but exacerbate the milder ENF phenotype of weak dominant negative numb mutants. Therefore, Dronc appears to provide an important but redundant function to limit neuroblast number.In the first example, apical caspase Dronc binds Numb to prevent the hyperproliferation of neuroblasts  Figure . As descdark or dronc as well as inhibition of caspase activity with a dominant-negative Dronc (DroncDN) or p35 in the scutellum produced extra SOPs and extra macrochaetes. One possible explanation for this phenotype is that caspases normally reduce SOP cell numbers through apoptosis. But this possibility was rendered unlikely by the absence of TUNEL-positive cells in the pro-neural clusters that produce the SOPs. Instead, caspases may suppress Wingless  signaling to limit the SOP number; a mutant allele of wg suppressed the extra macrochaetes phenotype that results from DroncDN while null mutants in Wg inhibitor shaggy  enhanced this phenotype. Sgg contains two caspase 3/7 consensus sites. Expression of Sgg with caspase consensus sites mutated produced extra SOPs and macrochaetes, leading to the model that caspases cleave to activate Sgg, which then opposes Wg to limit the SOP number (Drosophila I-kappaB kinase (IKK)-like kinase regulates the turnover of Diap1 to temper caspase activity and enables it to provide a developmental role without inducing cell death , which encodes for a signal transducer in the Hippo (Hpo) pathway, produced extra macrochaetes as did over-expression of transcription factor Yorki , an inhibitory target of Hpo signaling. The macrochaetes phenotype of ex mutants was rescued by reducing diap1, a known transcriptional target of Yki  that acts through Jun N-terminal kinase (JNK) and Mitogen-Activated Protein kinase (MAPK). NP can be visualized as preservation of severed dendrites following axotomy and is proposed to buy neurons time to maintain structure and function through the period of axon regeneration. RNAi against dronc or mutations in drice increased the fraction of dendrites protected in ddaE neurons, suggesting that these caspases normally contribute to the removal of severed dendrites, much like in pruning, and must be overcome for NP. The authors then tested the hypothesis that uncontrolled NP might interfere with subsequent axon regeneration. Indeed, neurons with dronc RNAi showed reduced regeneration, leading to the conclusion that Dronc activity promotes axon regeneration by limiting NP.A more recent study shows that apoptotic caspases play a non-apoptotic role in a phenomenon called neuroprotection that occurs during axonal regeneration . BrieflyDrosophila has parallels in mice where proteasome activity and localized expression of an IAP protein keep the activity of caspases 3 and 9 away from the cell body and restricted to axons to be pruned  or its genetic interactor spaghetti in Drosophila clock neurons led to Dronc activation as detected with an antibody against the cleaved form. Dronc activation in these experiments was observed only during daytime or after light exposure during the night, could spread to nearby non-clock cells, and increased with fly age. Expression of a dominant-negative dbt in the Drosophila eye led to Dronc activation in this tissue, cleavage of co-expressed human Tau in a Dronc-dependent manner, and neurodegeneration phenotypes. These results in Drosophila parallel the relationship between apoptotic caspase activity, Tau and neurodegenerative disease observed in mammals as described in the preceding paragraph. Furthermore, Drosophila studies add to this picture by identifying Dronc activation as a possible mechanism that links circadian dysregulation with susceptibility to neurodegeneration.A neration . DisruptDrosophila show that dying cells send mitogenic signals that promote tissue homeostasis. In Drosophila larval wing imaginal discs, Dronc promotes Apoptosis-induced Proliferation (AiP) through different mechanisms. First, Dronc acts together with JNK to produce Wg that acts as a secreted mitogen  where Dronc and Eiger function in a non-apoptotic manner to promote sensitization to pain . The latter outcome can be observed as an adverse response to thermal stimuli that would not elicit a response in non-irradiated animals. Dronc is required for both UV-induced apoptosis and thermal allodynia while effector caspases Drice and Dcp1 are required only for apoptosis, suggesting that Dronc plays a non-apoptotic role to induce allodynia. This idea is supported by the finding that doses of UV that are too low to induce apoptosis still induced thermal allodynia in a Dronc-dependent manner. Dronc, it was found, acts in the epidermis to produce Eiger/TNF that then signals through TNF Receptor (TNFR) on sensory neurons to induce thermal allodynia. Induction of Dronc in the epidermis, even when effector caspases are inhibited to prevent Dronc-induced apoptosis, results in thermal allodynia in an Eiger-dependent manner without UV exposure, further lending evidence to a non-apoptotic role for this apoptotic caspase.A parallel story has emerged recently in  to pain  (Figure  to pain . The expvia mono- or poly-ubiquitination (Drosophila development. An RNAi screen using a version of the CasExpress reporter identified genes that altered the number of living cells with past caspase activity (Given that activation of apoptotic caspases can lead to apoptosis, how do cells restrict this activity for other purposes without being killed? Two mechanisms emerge from the examples described here: keeping caspase activity too low for apoptosis or restricting it spatially. For both mechanisms, regulatory inputs appear to integrate at the level of Diap1, an E3 ubiquitin-ligase that inhibits Dronc tination . For thetination . These ractivity . Some of


Emotions are multi-component phenomena: relevant stimuli lead to automatic changes in multiple functional components including perception, physiology, behavior, and conscious experiences. Brain activity during naturalistic stimuli reflects all these changes, suggesting that parsing emotion-related processing during such complex stimulation is not a straightforward task. Here, I review affective neuroimaging studies that have employed naturalistic stimuli to study emotional processing, focusing especially on experienced emotions. I argue that to investigate emotions with naturalistic stimuli, we need to define and extract  A touching movie, a horror story, or a vivid discussion all evoke strong emotions. What are the neural dynamics of emotional processing in such natural conditions? Affective neuroimaging has traditionally used simple, controlled paradigms, that rely on varying one aspect of a stimulus at the time and often use short, prototypical emotion stimuli such as static images, movie clips, or facial expressions. Naturalistic paradigms have emerged as an approach in neuroscience with great potential for deciphering the neural dynamics in close-to-real-life settings  define emotion-related functional components of interest and emotion features reflecting fluctuations in them, (2) extract the emotion features continuously across the stimulus, and (3) integrate the features with brain imaging data to model brain responses underlying different functional components. The current review highlights how these challenges have been solved in affective neuroimaging so far.emotion feature refers to any emotion-related variable that we can continuously extract from either the stimulus or the observer  while annotations refer to features that are considered relatively consistent across individuals . Continuous emotion rating tools originate from the intensity profile tracking approach, which modeled emotion-related changes during stimulation using manual drawings . More reHuman annotations have been used to extract various emotion, semantic, and object features from naturalistic stimuli, ranging from low-level visual features to scene contents and portrayed emotions. Manual annotation systems of stimulus-related emotion features have been developed for different social features , emotionHuman ratings are currently the main method for accessing and modeling observer’s emotional experiences. Most naturalistic studies in affective neuroimaging have collected self-report ratings of some experienced aspect of emotions, including affective dimensions  encoding single voxels or regions corresponding to single or multiple features, (2) employing inter-subject synchrony measures to detect stimulus-induced brain activity, (3) decoding feature time series from the activity pattern time series of multiple voxels , or (4) extracting changes in functional connectivity related to different features.Encoding models in brain imaging use the stimulus to predict brain responses, while the complementary decoding models (see section “Decoding Models”) use the brain activity to predict information regarding the stimulus e.g., . In geneBy far, the most used approach for combining emotion features to brain imaging data is a univariate approach employing a general linear model (GLM) with parametric regressors , which provides a continuous measure of inter-subject similarity . ISPS coComplementary to encoding models, decoding models start with the entire pattern of activity across multiple voxels to predict information regarding the stimulus . EncodinFor instance, Finally, emotion features might also modulate functional connectivity. Once the network time series have been extracted, the methods for combining network time series with emotion features are the same as for voxel or region time series, including encoding, decoding, and inter-subject approaches. In BOLD-fMRI studies, functional connectivity is defined as stimulus-dependent co-activation of different brain regions, usually measured as a correlation between two regions’ time series. Thus, functional connectivity does not necessarily reflect the structural connectivity between two brain regions – two regions might not be structurally connected but still share the same temporal dynamics. In contrast, effective connectivity can be used to investigate the influence one neural system exerts over another .Network modulations of emotion features have been studied with functional connectivity methods including seed-based phase synchronization , and fronto-opercular  regions for valence and those of subcortical, cerebellar and frontocortical regions for arousal, while connectivity changes due to positive valence and low arousal were minor and involved mostly subcortical regions .Taken together, affective dimensions including valence and arousal are linked to wide-spread activity changes across the brain. Thus, parsing their effects in specific functional components by extracting and modeling other, more low-level emotion features is necessary for understanding what aspect of emotional processing the dynamic ratings of valence and arousal are measuring.Besides affective dimensions, another option for directly extracting the emotional experiences is to collect ratings of experienced intensity for various emotion categories, such as joy or fear. Based on previous results in multivariate pattern recognition studies, experienced emotion categories modulate brain activity across a wide range of regions , 2018. FMost studies with categorical emotions have focused on modeling the dynamic variation of intensity for one emotion category at a time. Thus, the stimulus materials in affective neuroimaging studies have often targeted one emotion category  and accumulation (i.e., profiles increasing over time vs. going back to baseline; Also, different emotional states might vary in their temporal dynamics. For instance, while changes in cortical arousal compared to baseline were visible for resting-state following emotional narratives evoking various emotions, their temporal dynamics differed from each other . Thus, dA central problem in affective neuroscience relates to ignoring individual differences. Often, studies have assumed that similar emotions are elicited in all participants, implying that the idiosyncratic part of the BOLD signal is uninformative of the phenomenon of interest. However, this averaging approach ignores the considerable individual variation in emotional experiences and in the functioning of different components, including emotion elicitation, bodily changes, and behavior. For instance, brain activity patterns underlying different emotions are more consistent within individuals than between individuals, demonstrating individual variation either spatially or in emotional processing in general . FurtherSimilarly, studies investigating individual differences in rating behavior have demonstrated subject-specific delays between an emotional event and its annotations , suggestOne way to accommodate individual differences is to model the time series individually. Individual models would need to cover quantitative and qualitative differences between participants and account for noise in individual ratings. For instance, Another approach to individual differences is investigating the time points in naturalistic stimuli when emotional processing either converges or diverges. For instance, pairwise synchronization calculated with ISC has been used to identify time points where autistic and typically developing individuals’ brain activity diverges during movie viewing . FeatureFinally, individual differences can be approached by identifying the regions which are differentially recruited across individuals . Inter-sNaturalistic stimuli provide an important step forward in bringing ecologically valid stimuli to laboratory conditions. Future research will likely move toward even more naturalistic conditions, such as modeling emotions during real-life interaction and for multiple people. These approaches are now becoming possible due to rapid methodological developments.However, the growing interest in naturalistic paradigms should always be guided by theoretical models . OtherwiAffective neuroimaging studies using naturalistic stimuli have focused on a few features at the time, or at least on features from a single or a few components. Relatively few studies have combined emotion features across functional domains. The lack of multi-feature approaches might lead to too simplified explanations of complicated phenomena . FocusinNaturalistic paradigms do not aim to replace the classic, controlled neuroimaging paradigms . Due to Finally, a word of caution is warranted: the definition of a naturalistic experience in the context of emotions requires consideration. The term ‘naturalistic’ originates from vision studies, where video clips correspond to naturalistic viewing conditions in contrast to static images. However, films and stories do not necessarily cause real-life-like emotions (see, e.g., HS conducted the literature review and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Indeed, simultaneous parallelization of the analysis of peptides and single cells is a promising approach for multiplicative increase in the speed of performing deep and quantitative single-cell proteomics. The community is ready to begin a virtuous cycle of increased adoption fueling the development of more technology and resources for single-cell proteomics that in turn drive broader adoption, scientific discoveries, and clinical applications.Single-cell tandem MS has enabled analyzing hundreds of single cells per day and quantifying thousands of proteins across the cells. The broad dissemination of these capabilities can empower the dissection of pathophysiological mechanisms in heterogeneous tissues. Key requirements for achieving this goal include robust protocols performed on widely accessible hardware, robust quality controls, community standards, and automated data analysis pipelines that can pinpoint analytical problems and facilitate their timely resolution. Toward meeting these requirements, this   •Wide adoption of single-cell proteomics can empower biology.•Protocols for single-cell proteomics by MS are ready for broad adoption.•Parallelizing the analysis offers multiplicative throughput gains.•Quantity controls and community standards are essential.•Single-cell proteomics offers unique perspective on biology. Single-cell proteomics will drive the next wave of single-cell biology. This requires broad adoption of existing methods, the application of rigorous quality control standards, and the continuous advancement of the technology. The advancement will be driven by numerous innovations, including highly parallelized analysis, and will increase the throughput, quantitative accuracy, and the accessibility of the single-cell proteomics. Thisin vivo samples. Indeed, even isogenic cell populations may exhibit significant biological heterogeneity , which prompts the question of when to use scRNA-Seq and when to use single-cell proteomics. The simple answer is to measure RNAs if interested in RNAs and measure proteins if interested in proteins. This simple answer is complicated by hopes that mRNA levels are reliable surrogates for protein levels . The degThus, instead of assuming that RNA levels faithfully reflect protein levels, we should measure both proteins and RNAs. Such joint measurements can reveal regulatory mechanisms. For example, covariation between the levels of transcription factors and mRNAs may suggest transcriptional regulation, whereas divergence between the RNA and protein levels of a gene may suggest post-transcriptional regulation of protein synthesis or degradation. Thus, joint single-cell proteogenomic analysis may enable characterizing both transcriptional and post-transcriptional regulation in single cells , 20, 21.via miniaturized sample preparation  making the approaches robust and widely available, that is, accessibility and (ii) increasing the number of cells that can be analyzed per project, that is, throughput. These requirements are discussed below, both their state and their prospects for further development.Ideally, any laboratory capable of performing quantitative MS proteomics should be able to perform quantitative protein analysis in single cells. Achieving this goal requires robust single-cell proteomics protocols that can be performed on widely available equipment . This reThe aim for robustness and accessibility has been a guiding principle in the development of the SCoPE2 protocol . SpecifiA major impediment to implementing MS methods can be the overhead associated with method optimization for each project, which may be very time consuming. This overhead may be reduced by highly detailed protocols that minimize the rediscovery of pitfalls. Nonetheless, even the best protocols tend to require some adaptation and troubleshooting. These aspects can be greatly facilitated by user-friendly computational pipelines that allow for quick diagnosis of problems and parameter optimization . Examplehttps://doi.org/10.5281/zenodo.4339954), its implementation in the scp Bioconductor package that offers increased functionality . This important next step should be established by an authoritative white paper articulating best practices and recommending quantitative benchmarks and data reporting formats.All data processing pipelines should transparently report quality control metrics based on consensus community standards . Such coHigh throughput is essential for many biological investigations, especially for achieving high enough statistical power . In the Just a few years ago, relatively few single cells could be simultaneously prepared for analysis , 52, 53,While nPOP uses only commercially available equipment and reagents, the equipment is expensive and not widely available . Thus, nAs the rate of robust sample preparation has increased, the rate of MS analysis of samples has become limiting. The two principal approaches to relieving this limitation are (i) increased multiplexing and (ii) decreased MS time per sample . IncreasMultiplexing single-cell proteomics can introduce batch effects and help mitigate them. Variability between batches of mass tags or tag-specific biases could result in batch effects. Such problems can be minimized by using high-quality isotopologous mass tags. Batch effects originating from multiplexing can be reduced by using reference samples to normalize for set-specific artifacts , 39. ExpThe second approach to increased MS throughput is decreased MS time per sample . Such deIdeally, throughput can be synergistically increased by combining short separation times and parallel analysis of both peptides and single cells . This coIncreasing appreciation for the need to perform single-cell protein analysis has propelled the field of single-cell proteomics by MS, resulting in methods that allow quantifying over a thousand proteins per cell while analyzing hundreds of single cells per day. Taking advantage of these capabilities requires their broad dissemination, which in turn requires robust and accessible protocols and data analysis pipelines. These requirements are already available to support the first wave of technology dissemination, and this dissemination will in turn drive the development of more analytical and computational tools. Central to the success of this virtuous cycle is a set of community standards that will ensure rigor in data reporting and interpretation. The stage is set for scaling up single-cell proteomics to the challenges and opportunities of cutting-edge biomedical research.The author has filed patents for single-cell proteomics methods.


Primary antibody deficiencies (PADs) are the most common primary immunodeficiencies (PIDs). They can be divided into the following groups, depending on their immunological features: agammaglobulinemia; common variable immunodeficiency (CVID) isotype; hyper IgM isotype; light chain or functional deficiencies with normal B cell count; specific antibody deficiency with normal Ig concentrations and normal numbers of B cells and transient hypogammaglobulinemia of infancy. The role of vaccination in PADs is recognized as therapeutic, diagnostic and prognostic and may be used in patients with residual B-cell function to provide humoral immunity to specific infective agents. According to their content and mechanisms, vaccines are grouped as live attenuated, inactivated , mRNA or replication-deficient vector vaccines. Vaccination may be unsafe or less effective when using certain vaccines and in specific types of immunodeficiency. Inactivated vaccines can be administered in PAD patients even if they could not generate a protective response; live attenuated vaccines are not recommended in major antibody deficiencies. From December 2020, European Medicines Agency (EMA) approved vaccines against COVID-19 infection: according to ESID advises, those vaccinations are recommended in patients with PADs. No specific data are available on safety and efficacy in PAD patients. Primary antibody deficiencies (PADs) are the most common primary immunodeficiencies (PIDs), representing, at least, 50% of all symptomatic forms . PADs caStreptoccoccus pneumoniae, Haemophilus influenzae and Gram-negative bacteria. These infections predominantly affect the respiratory tracts and, if untreated, lead to severe complications, such as chronic sinusitis and bronchiectasis. Less frequently, patients with PADs suffer from intestinal tract infections caused by Giardia spp., Campylobacter jejuni, Salmonella spp. or Helicobacter pylori and to bacterial cutaneous infections. Remarkably, agammaglobulinemic patients can suffer from severe, chronic enteroviral infections suggesting a major role for antibodies in preventing the dissemination of enteroviruses from the gut.Clinical manifestations include  autoimmuThe use of IgG replacement therapy (IGRT) represents the therapeutic cornerstone of all PADs. Substitutive therapy has changed survival and reduced the incidence of pneumonia and serious infections. Igs preparations include high titers of antibodies for many vaccine-preventable viral agents except for certain viral agents such as HPV and currently circulating influenza . DespiteThe role of vaccination in PADs has long been discussed, due to impaired antibody response and IGRT. At present vaccination is recognized as a therapeutic, diagnostic and prognostic tool in this patient population.Immunocompromised patients have an increased susceptibility to vaccine-preventable infections, even if vaccination is a controversial issue in this population ,7.Vaccination in PADs can be used in patients with residual B-cell function to provide humoral immunity to a certain infective agent and to improve the outcomes related to vaccine-preventable disease, as demonstrated by our group studies. It has also been proven that vaccination may induce some cellular immunity . In thisBonilla  states tThe gold standard to evaluate assessment of T-independent antibody responses, in clinical practice, is represented by the response to pneumococcal polysaccharide vaccines, and 23-valent pneumococcal polysaccharides vaccine (PPSV) is the most used. Antibody titers should be measured by the same laboratory before, and after 4 to 8 weeks from vaccination. The suggested IgG threshold for protection with respect to a single serotype after PPSV is 1.3 mg/mL . HistoriWall et al.  propose All these thresholds only take into account IgG response to immunization, while protective titers of specific anti-PPSV IgM and IgA are not defined at all.Systematic vaccination with PCV, recommended by the Center for Disease Control and Prevention (CDC) in childhood and in at-risk-group patient schedules, may limit the use of PPSV for assessing specific polysaccharide responses, due to a “priming” effect of PCV13 in enhancing a subsequent response to PPSV .Salmonella typhi pure polysaccharide vaccine (Typhim Vi) have been studied [In addition, patients on IGRT may present high titers of pneumococcal antibodies because preparations are rich in anti-pneumococcal IgGs. This raises the question whether to use specific IgA and IgM titers to evaluate the response. Alternative or complementary measurements of other polysaccharide responses have also been proposed, in an attempt to increase diagnostic accuracy. For example, responses to the less frequently scheduled  studied .Sanchez et al.  demonstrThe capability of CVID patients to be immunized through vaccinations has not only a therapeutic and a diagnostic role, but also a prognostic role. Our study group investigated the residual ability to mount an IgM and IgA anti-polysaccharide (PS) response in CVID patients under IGRT . Since aOn the basis of their antibody response after vaccination, patients were subdivided into three groups: group one, , non-responders, with low specific IgA and IgM; group two, , IgA and IgM responders; group three, , IgM-only responders. Group 1 had a significantly lower memory B cell frequency compared to groups two and three, suggesting a correlation between anti-PPSV IgM and IgA responses and B cell subset frequencies. We also found concordance between anti-PPSV IgM and IgA levels and clinical phenotype, since we noticed a higher prevalence of pneumonia and bronchiectasis in group one compared to group two or group three .We have also recently published data from a long-term observational study in the same population of CVID patients immunized by a single dose of a 23 pneumococcal polysaccharides vaccine . We collAs stated before, PAD patients may show a low or absent antibody response to vaccines; however, vaccination may induce some cellular response. On the other hand, vaccination may be not safe or may be less effective when using certain vaccines and in specific types of immunodeficiency . At presInactivated vaccines may be administered in primary antibody deficiencies even if they could not generate a protective response . Novel mRotavirus vaccines [In major antibody deficiencies  live attenuated vaccines are not recommended  but, accvaccines . Live atvaccines  and oralvaccines ,21 in pavaccines . Moreovevaccines . No datavaccines .Salmonella typhi live attenuated vaccines are also currently contraindicated in major PADs [BCG  and Salmjor PADs , despitejor PADs .Minor antibody deficiencies include selective IgA deficiency (SIGAD), specific polysaccharide antibody deficiency (SPAD) and isolated IgG subclass deficiency. Those patients should receive vaccinations as scheduled for a healthy population , althougAnnual vaccination against influenza virus, in particular, is recommended in PAD patients and, also in household contacts, due to the annual variability of the viral antigens .In the case of international travel, specifically required vaccinations should be individually evaluated. Rabies vaccine is an inactivated vaccine, safe in immunocompromised patients for pre-exposure prophylaxis in high-risk occupations and post-exposure to an infected animal, together with rabies immune globulin or when travelling to endemic areas. Yellow fever vaccine is a live attenuated vaccine and should not be administered to patients with severe humoral defects. It may be considered, in patients with minor antibody deficiencies, according to the risk–benefit balance. Inactivated Japanese encephalitis vaccine is expected to be safe, as are other inactivated vaccines .Pregnant women with PAD should follow the current vaccine recommendations regarding pregnancy and receive TDP and Influenza vaccinations, as these are safe in PAD patients ,27.According to the suggestions of the Medical Advisory Committee of the Immune Deficiency Foundation  and of tAn antibody defect may be also present in combined and syndromic immunodeficiencies. However, in these patients the vaccine schedule depends on the impairment of T cell function .In secondary antibody deficiencies the vaccination schedule is influenced by the underlying condition , that may be active or in remission, and by the eventually undergoing treatment . Vaccines containing purified antigens or inactivated organisms, including pneumococcal or influenza vaccine, are generally safe and not associated with increased risk of adverse events. Live vaccines are instead contraindicated or should only be administered after evaluation of the risk–benefit balance, during disease remission and after stopping chemo-immunotherapy . The assFor patients receiving IGRT, it is important to know when the last Igs administration was performed. Administration of IGRT should be separated from any other new drug administration, whenever possible, to facilitate the interpretation of any possible adverse reaction or symptom occurring in the hours or days following. Available Igs preparations contain a wide range of specific antibodies and may exert different antimicrobial effects. There is not a plausible rationale for why intravenous Ig (IVIg) should reduce the effectiveness of inactivated vaccines . ConsequSince February 2020 we have been experiencing a pandemic caused by a new coronavirus (Sars-CoV-2), causing COVID-19 disease, that has led to over two million deaths worldwide to date .Social distancing, the use of masks and frequent hand washing have become the cornerstone of the fight against COVID-19 disease. At present COVID-19 behavior in patients with inborn errors of immunity (IEI) seems not to differ from the general population, except for a younger age and a longer-lasting SARS-CoV-2 swab positivity. Most patients with antibody production defects do not experience severe disease, even though co-morbidity seems to be crucial in determining outcomes ,31.Since the beginning of the COVID-19 epidemic in Italy, most PAD patients have been informed about safety measures and shifted to home therapy and to remote assistance, in order to reduce the risk of infection . At pres+ T cell responses, due to the presentation of endogenously produced antigens on MHC class I molecules, in addition to potent CD4+ T cell responses and the generation of potent neutralizing antibodies.Starting from December 2020, the EMA approved Pfizer/Biontech  and ModeCurrently, no specific data are available on the efficacy and safety of COVID-19 vaccinations in patients with primary immunodeficiencies. However, in line with what is recommended for other inactivated vaccines, the ESID advises  that PADSince Jenner tested the smallpox vaccine in 1798, millions of lives have been saved. Vaccinations are a safe and effective tool preventing infectious diseases, not only by their direct effect on vaccinated subjects, but also indirectly, providing protection to unvaccinated people . Vaccines are also effective in the prevention of tumors, (HPV and HBV). Moreover, vaccines play an important role in PAD because they are useful for the diagnosis of antibody deficiencies while still being protective in patients with PADs. Considering that pneumococcal vaccination is increasingly entering into vaccination schedules, and due to the aforementioned evidence, S. typhi Vi vaccine may also be considered in the diagnostic work-up of PAD patients’ diagnosis. Moreover, IgA response to PPSV vaccine could be used as a prognostic marker in CVID patients. A vaccination schedule is currently available for patients with PAD: from this schedule live vaccines are excluded only in severe PADs. Unfortunately, no data are yet available on the efficacy of anti-COVID-19 vaccines in this group of patients, although they were included in the vaccination campaign.


Small extracellular vesicles (sEV), which are released to body fluids  by all types of human cells, may stimulate or inhibit the innate and adaptive immune response through multiple mechanisms. Exosomes or sEV have on their surface many key receptors of immune response, including major histocompatibility complex (MHC) components, identical to their cellular origin. They also exhibit an ability to carry antigen and target leukocytes either via interaction with cell surface receptors or intracellular delivery of inflammatory mediators, receptors, enzymes, mRNAs, and noncoding RNAs. By the transfer of donor MHC antigens to recipient antigen presenting cells sEV may also contribute to T cell allorecognition and alloresponse. Here, we review the influence of sEV on the development of rejection or tolerance in the setting of solid organ and tissue allotransplantation. We also summarize and discuss potential applications of plasma and urinary sEV as biomarkers in the context of transplantation. We focus on the attempts to use sEV as a noninvasive approach to detecting allograft rejection. Preliminary studies show that both sEV total levels and a set of specific molecules included in their cargo may be an evidence of ongoing allograft rejection. During the last decade, there has been a progressive increase in the number of solid organ transplantations. According to the Global Observatory on Donation and Transplantation (GODT) data, produced by the WHO-ONT , in 2019, there were a total of circa 163,141 solid organ transplants worldwide . T. T87]. TThe last (but not least) mechanism of allorecognition is termed a semi-direct pathway C. The prAcquisition of donor intact MHC molecules occurs via capture of clusters of donor-derived EVs that carry these MHC molecules on the vesicle surface. The donor-derived EVs are released directly by graft cells or by donor migrating DCs that reach the graft-draining secondary lymphatic organs (SLOs). This way rec-DCs, residing in the graft-draining SLOs, become cross-decorated with unprocessed donor MHC proteins. The individual rec-DCs cross-decorated with donor-derived EVs can present host MHC II molecules, loaded with donor allopeptides to CD4+ T cells through the indirect pathway. Simultaneously, the same rec-DCs present donor intact MHC molecules on the surface of EVs attached to directly alloreactive CD8+ T cells, via the semi-direct pathway. Additionally, CD4+ T cells can promote, through the indirect pathway, the maturation of MHC cross-decorated rec-DCs that subsequently stimulate the direct pathway CD8+ T cells . The concept of “MHC-cross decoration”, coined by Yewdell and Haeryfar in 2005 , along wMarino et al.  suggesteAntibody-mediated rejection (ABMR), collectively with TCMR, can take part in graft rejection at every stage of this process. In the hyper acute rejections, pre-existing antibodies become the primary trigger of endothelial damage and thrombosis. Inflammation and parenchymal damage visible in acute rejection are not only T cell-dependent processes, but are also associated with specific antibodies production. Acute and chronic AMBR can be triggered by memory cells in sensitized recipients, or by de novo generated DSA in nonsensitized ones . It has B cells may participate in graft recognition and rejection in two ways, either in cellular or in humoral reactions. In cell-mediated reactions, B cells primarily act as antigen presenting cells, thus leading to T cell activation. Alloantigen’s fragments, presented by MHC II molecules expressed on B cells, directly interact with primed Th2 cells in the indirect pathway of antigen presentation . The accDSAs, produced by activated B cells are considered a major reason of AMBR . FurtherThere are three major mechanisms through which DSAs induce reactivity towards the grafted tissue . FirstlyEven though B cells were confirmed as sEV producers in the late 1990s ,119, litImmunological tolerance is the absence of immune response toward specific antigens, but with preserved immune reactivity to others. This state is achieved through selective suppression of the immune reaction in specific settings. There are several ways through which the tolerance state can be induced. Central tolerance, generated in the immature embryonic stage of the immune system, provides non-response tolerance to the body’s own autoantigens. Therefore, it protects from unwanted reactivity of the immune system towards own tissues. According to the clonal selection theory, coined by Burnett as a consequence of the findings of Owens and Medawar, during the early development stage of the immune system a body can become tolerant to foreign antigens because of reactive lymphocyte clearance ,128,129.The immune homeostasis of a human body is also ensured by peripheral tolerance mechanisms that include clonal clearance, immunological ignorance, clonal anergy, immunological privilege, and the function of immunoregulatory cells . In tranExosomes are multipotent structures exerting diverse effects on target cells. In transplant immunology they act as a double-edged sword, either contributing to allorecognition and graft rejection or preventing the graft loss via induction of a suppressive phenotype of immune cells. Exosome-dependent allograft tolerance can be induced through several complex mechanisms, including split-tolerance and MHC cross-decoration. The phenomenon of split-tolerance and occurrence of non-inherited maternal antigens (NIMAs) is associated with maternal microchimerism (MMc). During pregnancy and breastfeeding a small number of maternal immune cells transferred to the offspring evokes tolerance to NIMAs in the latter ,141. MMcThymocyte release of sEV can influence the tolerance state through development and proliferation of T reg cells responding to TGFβ carried in EVs . ExosomeThe term “liquid biopsy” applies to the sampling and analysis of non-solid biological tissue, primarily blood. This type of analysis originally referred to diagnosis of solid tumors by means of harvesting and analyzing circulating tumor cells. This term was first proposed in 2009, during the Seventh International Symposium on Minimal Residual Cancer (ISMRC) in Athens, Greece. Later it was implemented in other clinical fields, such as transplantation. It was established that a graft can release exosomes into the bloodstream of a recipient. So far, exosomes have been studied in the context of heart, lung, kidney and pancreatic islets transplantations. Vallabhajosyula et al. were able to show specificity and reliable characterization of both pancreatic islets exosomes and kidney allograft donor exosomes for follow-up periods of five years . Sharma A special attention has been given to miRNA as a potential biomarker of the transplanted organ status. miRNAs are one of the most important types of non-coding RNAs contained in sEV. These short (ca.22 nucleotide) non-coding RNAs are known as molecules able to repress and degrade target mRNAs, thus silencing gene expression . miRNAs Exosomes have been extensively studied as a biomarker for transplanted lung rejection by Mohanakumar and colleagues. Gunasekaran et al.  analyzedIn 2017 Sukma Dewi et al. published a paper describing mi-RNA profiling in a group of 5 heart transplant (HTx) recipients with diagnosed acute rejection and 5 stable HTx recipients, and showed that exosomal miR-142-3p increased during rejection and augmented vascular permeability through down-regulation of endothelial RAB11FIP2 expression . Kennel 2 [In the context of KTx, studies have investigated RNA cargo and the proteome of exosomes from urine and plasma in search for potential biomarkers of acute rejection, both cellular and humoral, delayed graft function, interstitial fibrosis and tubular atrophy, calcineurin inhibitors’ (CNI) toxicity and non-specific kidney allograft dysfunction. In one of the first studies investigating urine exosomes in KTx recipients, the authors hypothesized that determination of NGAL in urinary exosomes would be a better predictor of kidney dysfunction after KTx than other urinary fractions . Indeed,2 . CandidaSo far, only a single study investigated exosomes in order to identify rejection and predict the outcome of liver transplantation. The authors analyzed the presence of galectin-9 protein in liver exosome cargoes from three sets of paired patients, with and without acute cellular rejection and in liver samples from 73 liver transplant recipients with rejection and 63 recipients with normal liver allograft function. Expression of galectin-9 was significantly higher in the TCMR group than in the non-rejection group, both in circulating exosomes and in liver samples. The recipients with higher liver expression of galectin-9 had worse survival .Exosomes have also been investigated as biomarkers of pancreatic islet allograft rejection in a human-to-mouse xenogeneic pancreatic islet transplant model. The authors isolated islet transplant exosomes from the recipients’ blood using an anti-HLA antibody. A significant decrease in the transplant islet exosome load and changes in the exosomal miRNA and proteomic profiles were detected prior to the appearance of hyperglycemia . During Over the past few years, sEV including exosomes have attracted great attention as a potential tool for examining transplanted organ/tissue physiology and disease. The utility of sEV has been investigated across various transplanted organs and various medical conditions including AR, using diverse biological samples, with focus on different elements of sEV cargo and with the use of several distinct techniques. In the case of lung transplantation EVs have been most extensively studied by Mohanakumar and colleagues who identified sets of surface molecules specific for BOS, AR and viral infections with the use of Western Blot technique. The researchers verified their results in small external populations, including pediatric patients. In heart transplantation, either miRNA or proteomic molecular signatures of AR were proposed based on sEV profiling. Neither of these were validated in external cohorts. In kidney transplantation, a number of potential molecular signatures of the most important both immunological and non-immunological causes of early and late graft dysfunction have been proposed. However, all were studied in small groups of cases, the results were inconsistent and none were validated in multiple independent cohorts. Only single studies have investigated sEV in the case of liver and pancreatic islets transplantation. Even though the published data seems promising, future studies are required before sEV analysis could facilitate clinical decision-making.Research on practical application of sEV is much hampered by an experimental setup where they form a heterogeneous population of vesicles released by various types of cells. This is the case, inter alia, in serum of patients after transplantation, where sEV released by donor’s cells are present alongside sEV produced by recipient’s cells. Small extracellular vesicles of lymphoid origin represent the largest group among all sEV identified in serum or plasma, which is a result of a large number of lymphocytes circulating in blood. It is estimated that sEV released by lymphocytes represent 60–80% of exosome population in serum/plasma, which poses an obvious problem when one aims at analyzing the composition of exosomes to be isolated from the general population of sEV. Furthermore, no standardized procedures for isolation of different EV types exist, leading to differences in contamination levels and co-isolation of various vesicles. The criteria and methods of EV characterization are also unclear and seem to change as we progress in understanding heterogeneity of EV. However, constantly improved sensitivity of instrumentation dedicated to analysis of sEV gives hope. Modern technologies enabling fast and sensitive detection of antigens present on the surface of sEV offer new opportunities of their monitoring. Nevertheless, future validation studies encompassing all aspects of sEV isolation, characterization, and signaling seem to be necessary to move the field forward and translate the current knowledge to clinically applicable strategies and methods. In this respect, antibody-based microarrays, multiparameter quantitative flow cytometry, and high resolution mass spectrometry-based proteomics are emerging as tools applicable to serial monitoring of sEV in body fluids of transplant recipients.


Extracellular vesicles (EVs) are subcellular carriers of bioactive compounds with a complex array of functional effects on target cells. In mammals, circulating bodily fluid microbiota EVs (mbEVs) deliver cargo from source cells and adversely or favorably alter the physiology of the same source, neighboring, and distant recipient cells in an autocrine, paracrine, or endocrine fashion, respectively. Plant mbEVs may similarly mediate these interactive effects within the holobiont framework. However, the majority of plant EV research has focused on a small number of individual microbes, thus failing to reflect the importance of EVs in a community and consequently leaving a wide gap in scientific knowledge. Addressing this gap should entail a systems-level approach that combines vesicle characterization with microbiome analyses. This would certainly usher in a new age in microbial biotechnology entailing EVs as a microbiome manipulation strategy, a biomarker for stable microbiomes, and a diagnostic tool for plant infectious diseases. One of the most pressing agricultural challenges of the 21st century is improving crop yield and quality. Efforts to address this challenge vary but often entail the use of microbiota . However, lifting the veil on microbial community dynamics to optimize plant production is not straightforward since diverse factors, including plant health and the environment, affect community composition. In turn, plant community relationships rely heavily on intricate systems for effective resource allocation and communication, one of which is embodied by extracellular vesicles (EVs).EVs are lipid bilayer-enclosed nanoparticles of about 30 nm to several micrometers in diameter. They facilitate the intercellular transfer of molecules from source cells over short and long distances, which are then absorbed by recipient cells via membrane fusion and endocytosis. EV-based communication systems are well understood in mammals, where their release can be deregulated in diseased but not healthy cells , suggestBecause EVs tend to mirror their donor cell features and functions, albeit to various degrees , they arArabidopsis thaliana and Nicotiana tabacum  mbEVs from endophytes colonizing the root apoplast can be isolated using protocols typically adapted for EV isolation from the leaf and root apoplast  it is not novel to find EVs during plant-endophyte interaction, and in fact, several ultrastructural analyses have detected late endosomal EVs at the plant-endophyte barrier , which iapoplast , 21; andapoplast . These mapoplast . Indeed,apoplast , highligExpanding upon the EV link between plants and their associated microbiomes for ultimately facilitating broad implementation of microbiome-based biotechnology in agriculture represents an intriguing opportunity. The extensive signal exchanges between microbial communities and plant host cells would certainly be reflected in community-released EVs. Therefore, integration of microbiome with EV studies would not only enhance microbial community profiling through molecular “omics” , but would also fuel a cascade of agricultural applications . This is


Leukocyte transendothelial migration is crucial for innate immunity and inflammation. Upon tissue damage or infection, leukocytes exit blood vessels by adhering to and probing vascular endothelial cells (VECs), breaching endothelial cell-cell junctions, and transmigrating across the endothelium. Transendothelial migration is a critical rate-limiting step in this process. Thus, leukocytes must quickly identify the most efficient route through VEC monolayers to facilitate a prompt innate immune response. Biomechanics play a decisive role in transendothelial migration, which involves intimate physical contact and force transmission between the leukocytes and the VECs. While quantifying these forces is still challenging, recent advances in imaging, microfabrication, and computation now make it possible to study how cellular forces regulate VEC monolayer integrity, enable efficient pathfinding, and drive leukocyte transmigration. Here we review these recent advances, paying particular attention to leukocyte adhesion to the VEC monolayer, leukocyte probing of endothelial barrier gaps, and transmigration itself. To offer a practical perspective, we will discuss the current views on how biomechanics govern these processes and the force microscopy technologies that have enabled their quantitative analysis, thus contributing to an improved understanding of leukocyte migration in inflammatory diseases. Leukocytes encompass a diverse group of white blood cells in the immune system, including lymphocytes, monocytes, dendritic cells, and neutrophils, which exhibit a versatile and broad range of migratory abilities. Leukocyte migration from the bloodstream to sites of injury or infection is a primary component of the innate immune and inflammatory responses. Functioning as first responders, leukocytes can efficiently overcome biophysical barriers in their response to pro-inflammatory stimuli, including the vascular wall and dense three-dimensional (3-D) extravascular spaces. This efficient pathfinding is essential for leukocyte trafficking and provides potential therapeutic targets for immune-related and inflammatory diseases.in-vitro and in-vivo experiments have demonstrated that the paracellular route is preferred, accounting for 90% of TEM events . The endothelium is formed by a monolayer of vascular endothelial cells (VECs) lining the vessel walls and functions as a physical barrier between the circulation and the underlying interstitial tissue. During TEM, leukocytes adhere to the VECs, transmigrate across the endothelium, and cross the vascular basement membrane to the extravascular space . Pro-infM events . It remaTEM involves several physical interaction cascades between leukocytes and VECs, characterized by the sequences of motions happening at the interfaces between the two cell types. Receptor-ligand interactions govern leukocyte TEM by modulating cellular functions, as mentioned above. For example, the activation of cell surface proteins triggers cytoskeletal rearrangements leading to increased cellular contractility and force transmission between the leukocytes, VECs, and the substrate. In this regard, TEM can be viewed as a biomechanically regulated process with contributions from both leukocytes and VECs. Recent advances in microfabrication, microscopy, and quantitative analysis allowed researchers to measure the mechanical forces involved in leukocyte-VEC interactions, contributing to delineating their roles in deciding the TEM route and driving cell motion. This review primarily focuses on two phases that play a determinant role in leukocyte trafficking: (1) adhesion and probing, and (2) direct TEM. Furthermore, we discuss current advances in force microscopy techniques for each phase and applications of force measurements in elucidating biomechanical mechanisms of leukocyte TEM. For additional background information on the biology of leukocyte TEM, we recommend previous reviews on this topic .in vitro and in vivo   and betwPodosomes are integrin-mediated adhesion structures observed in cells originating from myeloid tissue such as leukocytes and osteoclasts . These cThese results have raised the fundamental question of how leukocytes utilize podosomes and ILPs to mechanosense their microenvironment. Podosomes generate forces via their actomyosin apparatus and sense their extracellular environments via the integrin-based ring substructures in association with mechanosensitive proteins, which activate downstream mechanotransduction pathways to control various cell functions. This process can be utilized to probe substrate topographies, trigger extracellular matrix degradation, and sense the stiffness of the surrounding matrix or underlying endothelium.Studies on leukocyte adhesion to microfabricated substrates have found that leukocyte podosomes align themselves along substrate microgrooves . BecauseSuper-resolution microscopy has revealed that the F-actin podosome core is connected to a ventral F-actin module bound by vinculin and a dorsal module, crosslinked by myosin IIA and linked to other podosomes. Substrate stiffness influences the balance between these two modules allowing mesoscale podosome connections to collectively switch between the explorative, degradative behavior and the protrusive, non-degradative behavior . This stGiven that integrins are a primary structural component of the podosome ring, chemokines play an essential role in podosome formation by promoting the high-affinity state of leukocyte integrins . ImmobilVascular endothelial cells can modulate ILP activities by providing different ICAM-1 dependent ligand patterns , which cIn addition, ILPs display different characteristics from podosomes. In particular, ILPs on VECs have shorter lifetime than podosomes on extracellular matrices (seconds to mins vs. seconds to ten of mins) . FurtherAfter locating a hotspot on the endothelium, leukocytes shift from 2-D crawling to 3-D transmigration. Paracellular TEM is the most common route through the monolayer, mediated by the rapid disassembly of endothelial adherens junctions in response to an adherent leukocyte. The biomechanical interactions between leukocytes and VECs govern three crucial steps in this process. Specifically, mechanical forces contribute to opening endothelial gaps by destabilizing the junctions, help pull the leukocyte across the monolayer, and mediate the closure of the junctional gaps after TEM. This section discusses the currently recognized mechanisms and outlines open questions related to these three TEM steps.It has long been believed that VECs may play an active role in facilitating leukocyte TEM. VECs protrude microvilli-like projections perpendicular to the endothelium to form a specific “transmigratory docking structure” shaped like a cup, which can surround and hold an adherent leukocyte . These sThe anchoring and embracing functions of VEC docking structures are regulated by mechanosensitive ICAM-1-triggered signaling, including recruitment of actin-binding proteins, an increase in F-actin assemblies, and activation of Rho-ROCK pathways, all of which result in increased actomyosin contractility . F-actinBecause leukocyte sizes can be more than 20 times greater than the size of endothelial cell-cell junctions (∼10 μm vs. ∼0.5 μm), transmigration must involve precise biomechanical coordination between leukocytes and VECs. VECs actively respond to the leukocyte’s presence by forming gaps to accommodate paracellular TEM. The activation of endothelial ICAM-1 through leukocyte binding can trigger a downstream signaling pathway that promotes cytosolic calcium-mediated myosin activity, resulting in increased endothelial contractility. The resulting increase in the tensile force supported by the F-actin cytoskeleton  to the site of TEM . LBRCs aTo complete the picture provided by the above studies, one must consider that leukocytes are mechanosensitive cells that exert forces during migration and invasion . Recent Altogether, these studies show that junctional gap generation for TEM requires highly orchestrated biomechanical contributions from both leukocytes and VECs. Future studies are required to provide additional insight on exactly how these forces work together to promote leukocyte TEM.in vitro and in vivo did not affect the rate of leukocyte TEM events but caused leukocyte-induced vascular leaks. Moreover, a recent in vivo study found another Tie-2 receptor/Cdc42 GEF FGD5-stimulated mechanism responsible for preventing plasma leakage during leukocyte TEM. This study identified that platelets recruited to endothelial VWF activate the Tie-2 receptor by releasing Angiopoietin-1, reinforcing cable-like F-actin to close the endothelial pore  was developed as an extension of standard AFM to measure indentations made by cells seeded on a specially fabricated, compliant formvar substrate . The forWhile PFM provides invaluable information about the biomechanics of podosomes, it is a highly specialized technique. For instance, it is not straightforward to integrate PFM measurements of protrusive forces with long-term, whole-cell measurements of tangential traction stresses and motility. The emergence of elastic resonator interference stress microscopy ERISM,  now allo1/2 threshold is applied to the probe, opening the hairpin and (2) cyclic Arg-Gly-Asp-D-Phe-Lys (cRGD), which is localized on the upper arm and whose depletion is a proxy for podosome protrusive forces as measured by percentage decrease in fluorescence. Application of a force greater than or equal to F1/2 will open the probe, increasing both fluorescence intensity and lifetime  TFM, which measures the lateral traction forces parallel to the surface of cell attachment, was first applied by Dembo and Wang to fibroblasts migrating on flat substrates . This teIn micropost-based TFM, protein-coated arrays of microscopic pillars made from the deformable elastomer polydimethylsiloxane (PDMS) serve as a substrate . Cells aThe 2-D TFM is relatively straightforward on standard fluorescent microscopes, has excellent resolution, and can be adapted to a wide variety of applications . HoweverIn order to maintain homeostatic barrier function, VECs must regulate their monolayer tension to balance the biomechanical stability of cell-cell junctions and cell-substrate adhesions. This balance prevents cell adhesion forces from tearing the endothelium apart or detaching it from the substrate . During In comparison to AFM or TFM, the development of experimental, image analysis, and computational tools to quantify collective cellular forces has been recent. A salient technique is MSM, an extension of TFM that quantifies the collective distribution of intracellular stresses in thin confluent cell layers . Of notein vivo via deformable hydrogel microspheres could overcome this limitation. Inspired by the seminal use of functionalized oil droplets to measure anisotropic stresses within 3-D cell aggregates (An inherent difficulty in quantifying the biomechanics of TEM is to tease out the forces exerted by the leukocytes from those exerted by the VECs. To this end, ICAM-1 antibody-coated polystyrene beads mimicking firmly adherent leukocytes have been used in combination with TFM and MSM methods . Given tgregates , emergingregates . These eFinally, it is important to note that, although shear stress is an important regulator in inducing leukocyte TEM , most exLeukocyte recruitment is a hallmark of all acute and chronic inflammatory disorders. Understanding leukocyte TEM to inflammation sites could help identify therapeutic targets to boost immune defense and minimize inflammatory tissue damage. Currently, medical treatments of chronic inflammation employ general immune suppressors, which have numerous adverse side effects. This lack of success is attributed to the complexity of and the multitude of redundancies and interdependencies in the molecular pathways involved. Although many molecules, some of which are discussed above, have been implicated in TEM, their specific roles remain elusive. In particular, we still know little about how VECs and leukocytes orchestrate pathfinding and localized endothelial barrier modulation. These processes can depend on direct receptor-ligand biomechanical interactions and more intricate, collective mechanosensitive pathways. Their understanding requires advanced experimental techniques to detect subcellular, single-cell, and tissue-level deformations during leukocyte TEM, along with the corresponding development of computational models to analyze data streams of increasing complexity and size.in vitro assays close to reproducing physiological microenvironments under controlled conditions. In parallel, advances in 3-D imaging and force quantification have opened a window to data of unprecedented richness and quality. Indeed, these methodologies have significantly extended our current understanding of leukocyte trafficking. The current technological frontier is methods to allow investigation of these biomechanical events in vivo. Of note, emerging microscopy techniques such as two-photon, light sheet, and super-resolution microscopy will most likely play a transformative role in bridging the gap going from in vitro conditions to more realistic animal models. Heavy interdisciplinary efforts involving engineers, physicists, and mathematicians will certainly be required to overcome these formidable challenges.During the past ten years, the progress in the development of soft materials have brought AS, EC-H, SC, JÁ, JL, and Y-TY wrote the manuscript. AS and OC composed the figures and table. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.