Early introduction oral immunotherapy (E‐OIT) in the first year of life can be a safe treatment for infants with cow's milk allergy (CMA). Once the protocol is completed, doubts remain whether children achieve tolerance or remain desensitized. According to current guidelines, this is determined by an avoidance period followed by a re‐exposure to the food allergen during an in‐hospital oral food challenge (OFC). In real life, this approach can be complicated, time‐consuming, and anxiety‐provoking for parents. We assessed the long‐term safety of E‐OIT for CMA in a cohort of children who switched to an unrestricted diet without testing the achievement of tolerance at the end of the OIT protocol.We performed a descriptive analysis of the clinical follow‐up of a cohort of children diagnosed with IgE‐mediated CMA and undergoing E‐OIT protocol in their first year of life. In a previous publication, the same cohort of patients had been studied to assess the feasibility of E‐OIT for CMA. In the present study, we reported the results of a telephone survey, carried out through a questionnaire to their families enquiring about milk consumption and other ongoing atopic conditions of children.After an average of 4 years from the start of E‐OIT, 62/73 patients  participated in the survey. Among them, all 56 patients who had previously successfully completed the protocol reported an unrestricted cow's milk intake. Ninety–three percent of these children did not experience any further allergic reactions, while the remaining 7% described only mild and transitory reactions until the 6‐month period after the end of the protocol.This study confirmed the long‐term safety of E‐OIT for CMA and challenged the paradigm of the need for allergen food withdrawal to discern between desensitization and tolerance. It could be a starting point for planning future trials on this issue. The vast majority of these patients successfully introduced cow's milk (CM) along with dairy products in their diet, consuming them regularly over an average time of about 6 months and with no significant adverse effects.According to current guidelines, the acquisition of tolerance by OIT is defined as the lack of any allergic reaction after a period of avoidance followed by a re‐exposure to the culprit food allergen during an in‐hospital oral food challenge (OFC).Therefore, some doubts remain whether children achieve tolerance or remain only desensitized, once the protocol of E‐OIT is completed. Despite differentiating between desensitization and tolerance is considered crucial for identifying who is at risk for further allergic reactions, no studies have so far determined a desirable/required duration of the OIT maintenance phase to develop a state of sustained unresponsiveness which is not reliant on ongoing allergen exposure.From a pragmatic perspective, we suggest that a change of thinking in this approach could be considered outside of strict research protocols. Remarkably, CM is so widespread in western cuisine that an involuntary full and sustained avoidance is unlikely in real life.For this reason, in infants undergoing E‐OIT for CMA, we chose not to verify the achievement of tolerance through a period of CM avoidance followed by an OFC at the end of E‐OIT protocol. In this way, we targeted to reduce the risk of bringing patients back to a previous level of allergic reactivity and restarting oral immunotherapy from a milk dose lower than that previously gained.This study aimed to verify the long‐term safety of E‐OIT in a group of infants who had not been tested for tolerance at the end of the protocol but switched directly to an unrestricted diet.2We present a descriptive analysis of the clinical follow‐up of a cohort of infants diagnosed with CMA and undergoing E‐OIT in their first year of life, to assess the incidence of allergic reactions to CM at an average of 4 years from the start of the early introduction OIT protocol.2.1The same patients' cohort had been previously studied to investigate the feasibility of the E‐OIT protocol for the treatment of IgE‐mediated CMA in an uncontrolled longitudinal study.(i).A history of generalized allergic reactions in one or more organs  occurred within 2 h after CM ingestion.(ii).A skin prick test positive for fresh CM  and at least one of the main CM proteins between casein, beta‐lactoglobulin, and alfa‐lactalbumin .(iii).Specific IgE levels (ImmunoCAP® Phadia AB) for CM and at least one main CM protein higher than 0.35 kU/l.Were excluded from the study: children under three months of age, children with non‐IgE‐mediated clinical manifestations, and children with a known immunodeficiency.To test eligibility for OIT, infants were subjected to a low‐dose oral food challenge (OFC), with the administration of three increasing doses  of CM every 30 min. During the OFC, IgE‐mediated clinical manifestations were recorded according to Clark's classification.Every eligible infant followed a home oral immunotherapy protocol, with periodic increases of the milk dose, initially tested in hospital. Once the target dose of 150 ml of CM in the same meal was reached during the OIT, we taught parents to offer their children milk in quantities and varieties they liked, so that gradually reduced the need to offer milk regularly in pre‐established times and doses.Of the 73 children enrolled in the baseline study, 68 (93%) started the E‐OIT protocol: among them, 66 (97%) reached the CM target dose, that was a daily intake of 150 ml of milk or an equal dose of dairy, in a mean time of 6 months. These 66 patients, during the last clinical visit at the end of the protocol, were prescribed an unrestricted daily intake of CM or dairy products (given ad libitum). The remaining seven patients (10%) were not considered suitable for OIT or had to discontinue OIT due to persistent allergic reactions (mainly vomiting).The only atopic comorbidity of children screened in the baseline study was atopic dermatitis, which affected 58% of them.2.2In this long‐term follow‐up study, the whole cohort of patients was involved in a survey, in which outcome data were collected up to December 2019 through a telephone interview with their families.A follow‐up questionnaire, designed by researchers of University of Trieste, was administered to determine the current type of diet of children  and to investigate CMA recurrence after the E‐OIT protocol or CMA persistence without an E‐OIT approach.Information was also obtained about the evidence of other food allergies (still present or outgrown) and the presence of other atopic diseases.3A total of 62 out of the 73 patients of the historical cohort (85%) responded to the telephone survey, participating in the follow‐up study: children's median age was 4 years old and 68% were males. Among these 62 patients: five had not been passed through an E‐OIT attempt, due to generalized allergic reactions triggered by 1 ml of CM at the low‐dose OFC, while 57 had started E‐OIT. Of them, 56 had successfully completed the protocol, and one had dropped out for persistent reactions during the protocol.Figure All of the 56 cases (100%) who had completed E‐OIT reported an unrestricted CM intake. Based on the last 2‐week food diaries, these children consumed an average daily CM dose of 250 ml, equal to a daily intake of 8.3 g of milk proteins (~0.5 g/kg/die). Fifty‐two of them (93%) did not experience any further allergic reactions; the remaining four patients (7%) described CM‐related reactions until the 6‐month period after the end of the protocol. All these reactions were listed as mild and transitory, without need for medical intervention.The only child who had at the time dropped out of the E‐OIT protocol gradually reintroduced CM at home with no problems.Among the five children initially ineligible for OIT, three were still on an exclusion diet, while two reported an unrestricted intake of CM at the follow‐up: one of them after a second successful OIT, and the other one after a gradual CM introduction attempted at home by parents on their own initiative.Regarding the evolution of atopic march: atopic dermatitis persisted in 37% of patients (against 58% at the baseline), while other allergies (absent at baseline) developed in 16% of children. They were both food allergies  and inhalant allergies (mite and pollens).Table 4This study confirmed the long‐term safety of E‐OIT for CMA of other reports.Remarkably, by excluding from OIT children with generalized reactions triggered by 1 mL of CM, as well as possibly children with severe anaphylaxis at any dose, during the low‐dose OFC, we had selected a population of infants with CMA at low to moderate risk. In this perspective, E‐OIT, without a period of allergen absence, might mimic, in a sort of secondary prevention, what had happened in the cohorts of the studies that assessed the efficacy of early introduction of allergenic food in reducing the incidence of food allergy.This article limits were the lack of a randomized control group, a complete OFC at entry, a final dosage of cow's milk specific IgE and IgG4 levels.In any case, we aimed to report a pragmatic experience on a still little explored issue, concerning a significant number of patients and an extended period of follow‐up.Since we had excluded infants reactive to a very small amount of CM (1 ml), we could not extend our results to this population, theoretically at higher risk of severe anaphylaxis during OIT. Nevertheless, in Bonè Calvo et al.Further long‐term data are needed to confirm that tolerance achieved by early desensitized infants is as reliable as that achieved by children who spontaneously resolve their food allergy, and to define the adequate duration of OIT to move them safely towards an unrestricted diet. This report could represent a starting point for planning future trials on this issue.The authors declare that there are no conflict of interests.The Independent Bioethics Committee of the Institute for Maternal and Child Health‐ IRCCS Burlo Garofolo of Trieste  approved the protocol. All parents or legal guardians signed written informed consent for minors' participation.Laura Badina, Giorgio Longo, Irene Berti, and Egidio Barbi contributed to the article's conception. Giulia Peruch and Celsi Fulvio provided the data. Laura Badina, Laura Levantino, and Valentina Carrato wrote the draft and worked on the critical revision. All the authors read and approved this final version.


Malaria elimination will be challenging in countries that currently continue to bear high malaria burden. Sex‐ratio‐distorting gene drives, such as driving‐Y, could play a role in an integrated elimination strategy if they can effectively suppress vector populations. Using a spatially explicit, agent‐based model of malaria transmission in eight provinces spanning the range of transmission intensities across the Democratic Republic of the Congo, we predict the impact and cost‐effectiveness of integrating driving‐Y gene drive mosquitoes in malaria elimination strategies that include existing interventions such as insecticide‐treated nets and case management of symptomatic malaria. Gene drive mosquitoes could eliminate malaria and were the most cost‐effective intervention overall if the drive component was highly effective with at least 95% X‐shredder efficiency at relatively low fertility cost, and associated cost of deployment below 7.17 $int per person per year. Suppression gene drive could be a cost‐effective supplemental intervention for malaria elimination, but tight constraints on drive effectiveness and cost ceilings may limit its feasibility. The most commonly used vector control methods to prevent mosquito bites are sleeping under insecticide‐treated mosquito nets (ITNs) and spraying the inside walls of a house with an insecticide   WHO, . TreatmeRS) WHO, , 2019a, RS) WHO, . With 12Despite sustained malaria control, malaria incidence in the DRC has increased in the last few years WHO, , and morTransgenic mosquitoes carrying gene drives have recently been successfully developed in the laboratory  v2.18 IDM, , an agenWe selected eight provinces in the DRC for simulations Figure  in both An. gambiae mosquitoes, the only modeled mosquito species in this study, breed primarily in temporary puddles replenished by rainfall and drained through evaporation and infiltration  from the year 2000 to 2015 were retrieved from MAP rasters  by sampling global scale factors on the previously fitted seasonality profile to minimize the Euclidean distance between the model's parasite prevalence and the mean 2015 annual parasite prevalence of the location from MAP estimates who slept under an ITN the previous night: 6%, 38%, and 56% in 2007, 2010, and 2013, respectively, and applied to individuals of all ages.Case management of symptomatic cases with artemisinin‐based combination therapy (ACTs): 19% of uncomplicated malaria cases in all ages received treatment with artemether–lumefantrine, based on the 2013 DHS survey reporting 19% of febrile children under 5 receiving ACT.Indoor Residual Spraying (IRS) was not included as less than 1% of the DRC population was protected by IRS between 2007 and 2018 WHO, , 2019a.In the final 10 years of the 50‐year initialization period, the following interventions were imposed , and the fraction of offspring that are females is 0.5–0.5*(X‐shredder efficiency). Only females that mate with a driving‐Y male have their fertility reduced, and the total egg batch size is reduced by the fecundity reduction for each female that mates with a modified male . We did not include human migration in the simulations and found no difference in parasite reduction outcomes upon including human migration Figure .2, likelITNs at 50% coverage and ACT at 19% coverage were applied uniformly across all nodes in baseline scenarios, which were used as the main comparator against other scenarios. Each of the individual ITNs and ACT (case management rate with ACT) and the combination of ITNs and ACT was analyzed at three levels of coverage: 50%, 80%, and 95%, following the selected coverage levels in Evans et al. . If not We calculated Disability‐Adjusted Life Years (DALY) from model outputs—population by age group, uncomplicated clinical cases, severe cases, and deaths—at Year 5, Year 10, and Year 15 by giving equal weights to years of healthy life lost at young ages and older ages and with 0% discount rate for future lost years of a healthy life. The standard life expectancy at the age of death in years, and the DRC’s country lifetable WHO, , and disCosts of all interventions, including gene drive mosquitoes, were calculated standardized to the year 2000 adjusted for inflation for the results to be comparable to those of previous WHO milestone studies  eliminated malaria. In lower‐transmission settings, drives with slightly lower X‐shredder efficiencies (0.9 to 0.95) could also eliminate malaria. These parameter ranges resulted in the elimination of vector populations. At X‐shredder efficiencies where malaria elimination was not observed, the impact on adult vector population size was larger than the impact on parasite prevalence . In contrast, elimination was not achievable in Kwango, Nord Ubangui, and Kasai Central at these coverage levels, showing the need for new tools and echoing conclusions of recent eradication evaluation commissions , a hypothetical unit of currency that has the same purchasing power that the U.S. Dollar has in the United States at a given point in time (Tan‐Torres Edejer et al., 4This study uses mathematical modeling to describe the potential role of sex‐ratio‐distorting gene drive mosquitoes in malaria control across the transmission spectrum in the DRC, an area where achieving effective control has historically been challenging. Our results suggest that population suppression through gene drives could be an effective strategy for malaria elimination in the DRC, either as a single intervention or in combination with other interventions. To the best of our knowledge, this is the first study that models the epidemiological impact and cost‐effectiveness of gene drive mosquitoes for malaria elimination. Previous studies involving gene drives for malaria control are limited in scope to laboratory experiments (Akbari et al., Aedes and Culex mosquitoes (Burt & Crisanti, An. gambiae (Galizi et al., We found that the success of driving‐Y gene drives in all areas regardless of vector density highly depends on the ability of gene drives to shred the X chromosome. Though a naturally occurring driving‐Y chromosome that transmits >90% of male progeny can be found in Some sex‐ratio‐distorting drives may not be comparable to Y drive or X shredders, for example, if the female carries a drive that inactivates the reproduction of her male progeny. However, the difference in mechanisms by which sex ratio distortion is achieved may only lead to differences in how quickly a drive establishes itself rather than downstream outcomes regarding elimination, which is the focus of this study. The models predict that reaching malaria elimination does not always require bringing the number of mosquitoes down to zero and that there is continued mosquito biting after releasing gene drive mosquitoes. Our finding that the drive must be highly effective while mild fecundity costs are well‐tolerated is likely generalizable to other suppression drives as a whole, although further explorations in settings of stronger seasonality are needed. Our economic findings on the cost‐effectiveness of drives are likely to be order‐of‐magnitude similar for any highly effective suppression drive.The success of suppressive gene drives such as driving‐Y depends on mosquito population size and allowing enough time for the drives to propagate in the mosquito population. Understanding interactions between existing vector control methods such as ITNs and IRS that temporarily reduce the mosquito population (Alphey et al., Our models predicted that high coverage with ITNs and high access to treatment with ACTs could eliminate malaria in lower‐transmission settings, but achieving such high coverages of existing measures is not only extremely difficult but also comes with high implementation and logistical costs (Shretta et al., Our model results show that tailoring the frequency of releases and the number of gene drive mosquitoes to be released can make malaria elimination achievable within 5 years (Supporting information Wolbachia‐infected mosquitoes per person (Meghani & Boëte, We based our cost‐effectiveness analysis on the unit costs of OX513A (Alphey et al., Wolbachia‐infected mosquitoes, where costs came from expenses for the entire deployment (O’Neill et al., Wolbachia postdeployment would also reduce ongoing costs. The upper bound cost is from studies on the Oxitec mosquito (Meghani & Boëte, The lower bound cost applied in this study is from a study on Gene drive technology is at an early stage of development and concerns over ethics, safety, and governance, as well as questions on affordability and cost‐effectiveness, must be addressed before implementation WHO, . For higAn. gambiae, the predominant malaria vector in Africa (The Anopheles gambiae 1000 Genomes Consortium, An. gambiae in this study. The study identified key aspects of both gene drive technology and its implementation that are fundamental for the technology to be a cost‐effective component of a malaria control program. Amid uncertainty about vector abundance and its behavior (Guerra et al., This study demonstrated a modeling approach applied to The authors state that there are no competing interests.Supplementary MaterialClick here for additional data file.Table S7Click here for additional data file.


Our aim is to present the state of the art and identify challenges and gaps in current uEV‐based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast‐growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This  Early studies focused mainly on cancers related to the urogenital system and led to the identification of protein, mRNA, miRNA, lipid and metabolite biomarkers for prostate, bladder, and renal cancers , and utero‐vaginal tract  Table  Gonzale. The bioApart from being produced by different cell types in the urogenital tract, uEVs can also originate from residing immune cells, bacteria, and yeast, while enveloped viruses, themselves a type of EV, may also be present   detected 295 proteins . These EVs showed a remarkably high content of cholesterol (63%), with phosphatidyl serine 18:0/18:1 being the next most abundant lipid species . Within ongoing and future urine biobanking studies, we consider that special focus should be put on method validation/consistency and particularly on identifying the most and the least variable preanalytical parameters that affect EV research  and the investigated health condition or disorder. As long as standard operating procedures for collection and storage of uEVs are not established by the community, it is safest to report all available pre‐analytical information related to the studies in the EV‐TRACK knowledgebase, in accordance with the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) and other ISEV rigor initiatives as well as other suited guidelines developed particularly for preanalytical variables of fluid samples  or can be collected across several voiding episodes during a fixed time period (“timed urine collection”). Spot urine collections can be done at a random time (“random” spot urine) or standardized to the first or second morning urine. Timed urine collections can be over the course of hours or a day . The volume of urine collection can be “full void” or “midstream urine” . Relatively little is known about the impact of different collection types on uEV measurements.et al. found only minor differences between first and second morning urine with respect to total protein in uEVs or exosome‐associated proteins  by the urologist. Collection at this time point can greatly increase the amount of prostatic fluid in the urine and consequently enriches the sample for prostate‐derived EVs  particles. Models with a lid are preferable to prevent the introduction of external EVs. Sterile tubes may be especially important for studying microbial uEVs. Specialized collection devices might be needed  or part of a protocol for standardized collection of different voids .et al. found that miRNA and other small RNA contents of uEVs declined during 4–24 h of storage after collection, and the decline was greater when samples were kept at 2–4°C rather than at room temperature (RT)  and cooling (either to RT or +4°C) of the urine specimen during collection are lacking. However, generally, if long urine collection times are required, the addition of preservatives such as azide should be considered to avoid microbial overgrowth, at least when the preservative is compatible with further uEV processing steps   is generally used to remove whole cells and large cell debris, but data has shown that lower speeds may not suffice for this task. A single 400 × g step for 5 min results in inadequate removal of cells while efficient cell pelleting was achieved by centrifugation of 10 ml volumes at 1358 × g for 10 min in round bottom tubes  and the MMI Guidelines for Standardized Biobanking (crdi.ie/resources/biobanking‐guidelines/) from the Clinical Research Development Ireland. According to these sources as well as other uEV literature, centrifugation parameters used for preclearing vary widely. For example, the centrifugation speed used ranges from roughly 1–20,000 × g spin for 30 min without a gross loss of uEVs, whereas speeds ≥ 10,000 × g result in pelleting of uromodulin and EVs  for reproducibility in proteomic and metabolomics studies with the criteria that the urine supernatant should still contain the EV component  in urine samples stored at room temperature, at +4°C, or frozen for 2 h to 1 week , a well‐established cohort of urine samples with different levels of albuminuria, many isolated uEVs were associated with antigens including proteases, protease inhibitors and ubiquitin   that leak into the urine in glomerular disease can bind to the surface of EVs or co‐elute as a protein complex , and the presence of blood cells and lymphocytes in the urine samples. A gel electrophoresis can also show the protein pattern of each urine sample before and after uEV enrichment. This information can be considered during sample inclusion as well as when analysing the uEV fraction. Electron microscopy can be used to detect the presence of abnormal vesicle morphology or other structures in the uEV sample, such as uromodulin (precipitates). Western blot can be used to detect specific co‐isolating proteins that may be present in the EV sample such as uromodulin or albumin. Proteomics analysis is also useful as it allows to compare the abundance of these and other co‐isolating proteins in relation to uEV proteins.In conclusion, we note limited consistency and coherence between uEV separation methods. These limitations also apply to methods for characterization and analysis of EVs (addressed in the next section).3.33.3.1Authors reporting uEV characterization should be guided by MISEV reporting requirements , cryogenic electron microscopy (cryo‐EM), atomic force microscopy (AFM) and super resolution fluorescence microscopy , based on the Coulter principle . Western blot or ELISA techniques are based on bulk analysis of uEV content, whereas flow cytometry offers high‐throughput single‐EV surface protein analysis but requires advanced instrumental setup and experience to obtain sufficient resolution  being a major factor in the choice of isolation method. An interactive web‐based application (miRDaR) with incorporated comparative statistics was also developed to help investigators select the optimal RNA isolation method for their studies  and a subsequent protein fractionation step followed by mass spectrometry was recently described and applied for biomarker discovery in nephronophthisis‐related ciliopathies . Overall, urine analytes are relatively dilute and few platforms are sensitive enough to perform analysis without any pre‐enrichment processing, but quantification and characterization techniques developed for the analysis of cell‐depleted urine are making important progress, and might someday facilitate clinical application of uEVs.One of the best‐defined techniques for direct quantification and characterization of uEVs is NTA, which can measure particle size distribution and concentration in biofluids  in which a uEV marker is excreted. This can be measured using a timed collection, or may be approximated by normalization to urine osmolality or creatinine in a spot urine  per sample. After acquisition, protein data can be processed using quantile normalization, which assumes that the majority of proteins present in the sample are stable. Protein variation in uEVs has been recently determined by Oeyen, et al.  that is considered to be stable in the studied condition has been also proposed  is considered the gold standard to determine excretion rate of general urinary biomarkers such as albumin, because it is less sensitive to fluctuations due to circadian rhythm  can be used as a measure of the amount of prostatic fluid released in the urine and as a surrogate marker and normalization factor for the number of prostate‐derived uEVs  , keep samples cold  and consider additives  to avoid microbial growth and maximize preservation of the EVs.Gather dipstick data to indicate the presence of interfering or abnormal components.Centrifuge urine before freezing to remove cells that could be disrupted during freezing. Aliquot samples according to future use and available space. Freeze at ‐70°C or colder.Use only hygienic collection devices, containers and plastics that resist urine pH and do not bind uEVs (lipids/proteins) or shed particles.It is also important to collect and report low evidence level items to improve our understanding of the impact of these factors and reduce current uncertainties. These may include: Need for light protection or for some sample protecting agents, such as RNase inhibitors or cryoprotectants. Freezing speed. Quick freezing appears to work, but tests for a range of freezing speeds are lacking. Defrosting temperature.4.2As with most body fluids, urine contains EVs from a plethora of different organs, tissues and cell types from the urinary tract Figure . Togethetype of analytical parameter that is going to be studied. uEV analysis can be focused on physical parameters  and/or the biochemical content of uEVs . This is reflected by the wealth of state of the art and newly emerging EV assays and analysis technologies available  to confirm the presence of EVs  analyses to avoid interference of non‐EV contaminants in urine, but might be nonessential or maybe even disadvantageous for other analyses as any isolation or purification protocol unavoidably leads to significant loss of EVs and EV material. In addition, isolation procedures are generally biased towards certain EV size and density ranges. It is therefore recommended to avoid EV isolation or purification protocols as much as possible  capture of specific uEVs of interest  can enhance the specificity and sensitivity of the analysis. Such capture within the analytical assay relies on the availability of suitable capture targets on the EV surface and the efficiency of capture. Moreover, the yield of specific uEVs in these capture approaches could be a concern. Capture based assays often use the  general and abundant EV surface markers CD9, CD63, and CD81 for capture, for example, time‐resolved fluoroimmunoassay (TR‐FIA), surface plasmon resonance imaging (SPRi), ExoView®   EVs require individual samples be measured independently. Large‐scale experiments and studies on larger cohorts of uEV samples will require more high/medium throughput technologies. To support the scalability of uEV analysis, several technologies are being developed that enable higher throughput using automation and miniaturization of assays in (microfluidic) devices. Related to scalability is standardization. At this moment, many of the analytical assays for EVs are highly dependent on details in the protocols and settings. It is therefore pivotal to introduce optimal levels of standardization and reporting in the analysis of uEVs to improve reproducibility  – absolute excretion rate Creatinine/osmolality normalization – estimate of absolute excretion rate using spot urine Constitutively expressed uEV marker – relative excretion rate Specific marker ratio  – relative excretion rate Relation to total uEV count – relative excretion rate Z‐normalization (RNAseq/MassSpec) ‐ relative excretion rate To GFR (or nephron number) – relative excretion rate (organ‐related: kidney) Relation to PSA  – relative excretion rate (organ‐related: prostate)The strengths and limitations of each normalization method are mentioned in Table Important criteria for developing new normalization tools are: Decreases variation within normal or expected range Widespread availability and feasibility Can be validated internally and across testing sites, ideally with (shared) external standards Compatibility with commonly used isolation and/or analysis methods.4.44.4.1Common issues and general recommendations to be considered for attributing a functional activity to EVs are extensively detailed in MISEV 2018  enzymatic degradation of proteins or RNA/DNA species to allow investigations addressing the “EV‐corona” , urine presents certain specific challenges that must be considered when evaluating the functional activity of urine EVs. As detailed earlier, the timing and type of collection method may lead to dramatically different levels of cellular elements, including EVs in urine samples. Thus, when possible, the same collection method should be used for any comparative analysis. In addition, uromodulin can entrap small EVs in polymer “nets” and reduce recovery. Releasing EVs from uromodulin is therefore necessary to avoid a biased functional analysis which focuses on a small subset of urinary EVs. However, complicating this is the fact that procedures which disrupt the uromodulin network and release EVs  derived from epithelial cellsuEVs are (mostly) derived from three major organs: kidney, urothelium, prostateNormally, urine does not contain platelets or lipid particles other than EVsUrine has variable contamination with microbiotaUrine composition is highly variable  and influenced by certain medications and dietCollection:Urine collection is minimally invasiveUrine can be collected in large quantitiesUrine collection is sensitive to collection errors by the patients, that is, mid‐stream versus first void; incomplete timed collections, and so forthRelease of prostate EVs can be stimulated by digital rectal examination (DRE)Urine dipstick may be used as an easy quality control of urineUrine can contain cells that should (and can easily) be cleared before freezingSeparation / characterization:Uromodulin lowers yield of uEV separation techniquesKidney disease can cause proteinuria/albuminuria and interfere with molecular uEV analysisNormalization:An absolute uEV excretion rate can be determined from timed urine collection


It has not only led to a tragic loss of life and terrible economic costs but has also been met with an unprecedented response of the scientific and medical communities. In an effort to better understand this viral infection, scientists around the world generated the largest surge in research in documented history for any topic . A part of this work has included the need to better understand the impact of the virus on human proteins—the key machinery of the cell—and human physiology. In their recent study, Geyer and colleagues  that analyzes the plasma proteome changes for a longitudinal cohort of COVID‐19 patients.D. Memon, I. Barrio‐Hernandez, and P. Beltrao discuss the study by Geyer  In the context of cell‐based models, this technology has been applied to study how SARS‐CoV‐2 takes control of its target cell in order to identify potential host targeting drugs that could inhibit viral replication . . et al, et al, In addition to comparing COVID‐19 patients with controls, the authors studied how the plasma proteome changes along the time course of infection. This revealed broad trends of decreased abundance of innate immune‐related proteins; increased abundance of lipid homeostasis proteins and coagulation factors; and a more complex dynamical pattern of increased followed by decreased coagulation‐related proteins. When comparing 25 patients that survived with 6 that did not, they identified a small number of proteins that may potentially serve as biomarkers of disease severity, including the pro‐inflammatory acute phase protein ITIH4 and coagulation‐related proteins such as heparin cofactor 2 (SERPIND1), plasma kallikrein, (KLKB1), and plasminogen (PLG). The markers related to inflammation and coagulation were also independently identified as predictive of patient survival in an independent longitudinal plasma proteomic study of COVID‐19 patients (Demichev This study demonstrates how plasma MS‐based proteomics can be used to study relevant aspects of disease progression and to identify relevant biomarkers of disease and possibly also disease severity. Together with previous studies, this application to COVID‐19 further reinforces the importance of using these technologies as a tool for routine health assessment supplementing the blood tests already used. The findings from the longitudinal study argue for the importance and perhaps even the necessity of monitoring the plasma proteome over multiple time points during disease progression. The availability of plasma proteomics across multiple cohorts could be used in the future for meta‐analysis, which could identify the most robust patterns of protein changes and biomarkers.et al, This study raises questions related to the future implementation of these approaches, including the use of machine learning in a clinical setting, cross‐platform compatibility, and data access Fig . As this


BLC and RS were employed by Sinai Health which received funding from Seqirus for this review. MDMH and IM are employed by Seqirus.Brenda Coleman: Data curation; formal analysis; investigation; methodology; project administration; resources; supervision; validation. Ruth Sanderson: Data curation; investigation. Mendel Haag: Conceptualization; methodology; project administration. Ian McGovern: Conceptualization; data curation; methodology; project administration; visualization.https://publons.com/publon/10.1111/irv.12895.The peer review history for this article is available at Dear Professor Cowling,We would like to thank you for the opportunity to address the suggestions raised in the letter by Yin et al. in response to our study “Effectiveness of the MF59‐adjuvanted trivalent or quadrivalent seasonal influenza vaccine among adults 65 years of age or older, a systematic review and meta‐analysis” that found that among adults ≥65 years, aTIV demonstrated significant absolute vaccine effectiveness (VE), improved relative VE compared to non‐adjuvanted standard‐dose TIV/QIV and comparable relative VE to high‐dose TIV.The decision to focus on real‐world evidence was based on an interest in synthesizing data on the performance of aTIV/aQIV under real‐world conditions in the general population. Randomized controlled trials (RCTs) are generally considered the “gold standard” of evidence due to their potential to produce relatively unbiased effect estimates. However, clinical trial inclusion/exclusion criteria may result in a study population that is not representative of the general population, potentially decreasing the generalizability of the results to routine care in the general population. Seasonal influenza vaccines are reformulated almost every year due to the constantly changing antigenic properties of circulating influenza viruses. This frequent change necessitates annual evaluation of influenza vaccine performance, which is accomplished primarily though RWE.One study in our review  are frequently included in systematic reviews because it helps to ensure a comprehensive assessment of all available literature and the exclusion of that data can lead to publication bias.There were additional studies evaluated in our review that were not captured in the reviews conducted by Canada's NACI, the ECDC, or STIKO, predominantly due to the later search cutoff date for our review.The results of our study represented the most up to date body of RWE evidence related to aTIV/aQIV effectiveness (at the time of the review) and give insights into how the aTIV performs in a real‐world setting and population. The potential limitations inherent to observational evidence were acknowledged and discussed in the manuscript. Accounting for the various scenarios suggested by the responding authors did not impact the study's overall conclusion that among adults ≥65 years, aTIV demonstrated significant absolute vaccine effectiveness (VE), improved relative VE compared to nonadjuvanted standard‐dose TIV/QIV, and comparable relative VE to high‐dose TIV.


Within Australia, chronic silicosis has been long thought of as being a well‐controlled occupational lung disease. While recent cases of acute silicosis in artificial stone benchtop cutters have emerged, chronic silicosis within the general workforce population has not been recorded. Our case series describes the re‐emergence of chronic silicosis amongst workers within the tunnelling industry representing the potential for a more widespread insidious occupational lung disease. While undertaking pre‐employment medicals, eight tunnellers have been diagnosed with chronic silicosis. These eight tunnellers had a minimum of 10 years of cumulative dust exposure prior to diagnosis. Diagnosis was made by radiological evaluation of chest X‐rays and computerized tomography scans by International Labour Organization B Readers. The re‐emergence of chronic pneumoconiosis as illustrated by this case series suggests the presence of undiagnosed occupational lung disease with far reaching implications for workers, employers, compensation systems, and the public healthcare sectors. This article outlines the emergence of chronic silicosis in a population which has been previously thought as not being at risk for pneumoconiosis. It also outlines the concerns around fragmented reporting and compensation systems. Silicosis is a well‐recognized interstitial lung disease which has been described as early as 430 BC by Hippocrates . All typSilicosis is a spectrum of disease that is dependent on the cumulative dust exposure over time. Acute silicosis is associated with significantly high dust levels over short periods of time with a poor prognosis and in most cases mortality within five years. Chronic silicosis has a longer latency period of greater than 10 years. While chronic silicosis is initially asymptomatic, as the disease progresses, a worker may develop chronic obstructive airways disease, congestive cardiac failure, and be at increased risk of other diseases such as tuberculosis and lung cancer [3 (8‐h time‐weighted average) [In February 2020, new guidance around medical surveillance for workers exposed to respirable crystalline silica has been issued by Safe Work Australia , 6. ThisAn analysis of pre‐employment medical examinations for those workers within the tunnelling industry, between December 2019 and December 2020, was undertaken. These medicals included occupational and medical histories, spirometry, and chest radiographs read by the International Labour Organization (ILO) B readers. Initial review of the pre‐employment medical examination results was undertaken by occupational and environmental medicine physicians. Initial screening was first conducted by using ILO chest radiographs with findings confirmed with a computerized tomography scan. All identified cases were also referred to respiratory physicians for evaluation of the lung disease severity. Standardized lung function testing was performed.During this period, the review of 284 pre‐employment medicals was undertaken for persons exposed to silica working underground in tunnelling environments. This initial review identified 23 workers with comments on chest radiograph. Further internal review identified 11 workers with confirmed chronic silicosis. While 11 workers were confirmed with chronic silicosis, eight workers provided informed consent. Another worker was identified as having a false‐positive result, that is, the worker was thought to have radiological features on the initial ILO B read but was confirmed as negative by subsequent review of the ILO chest X‐ray and further computerized tomography scanning. The false‐positive case has been excluded from this case series.Our case series of the eight workers comprised of only males with an average age of 49 years (range 40–62 years). All individuals identified in the case series had a history of working on large infrastructure and construction projects across Victoria and New South Wales before travelling to Queensland to commence work on projects. While there were various job descriptions within the eight cases, all cases had been working underground within tunnels as they were being constructed. The average occupational cumulative dust exposure time was 21 years (range 10–44 years) with two workers having a mineral mining history of greater than five years in conjunction to their tunnelling experience. All workers reported undergoing pre‐employment medical surveillance conducted for other jobs over the last 10 years. Workers from New South Wales also reported undergoing periodic medical surveillance while employed on projects in New South Wales.The worker with the longest history of exposure (44 years) had been formally diagnosed and participated in an ongoing medical surveillance through a state‐based compensation system programme. In 2019, he had a reported ILO read of 2/3 with progressive massive fibrosis and was considered as fit to continue working with no advice being provided about suitable workplace control strategies. One worker had a differential diagnosis of silicosis in 2014 but was never followed up with our review providing him with the first formal diagnosis. Another worker had an ILO chest X‐ray of 1/0 with s/s nodules noted in 2019. No further investigations were performed at the time with the tunneller having been subsequently confirmed as 1/2 p/q in 2020.The remaining five workers were not aware of any diagnosis of silicosis being made prior to our review. The radiological features of a worker with 30 years of dust exposure has been illustrated in Figure From a clinical perspective, all workers were asymptomatic for features of chronic lung disease. Two tunnellers had radiological evidence of emphysema with another tunneller also having features of lung atelectasis.Attempts were made to source previous pre‐employment and periodic medical surveillance records for comparative analysis. However, as this information had not been directly provided to the workers and was gathered during employment for various other companies, attempts were unsuccessful. Information about personal protective equipment was also difficult to gather. Anecdotally, all workers especially those with longer exposure histories stated that workplace controls and personal equipment were limited on previous jobs. The reported anecdotal trend was that personal protective equipment had become more common over the last 10 years but engineering control still remained limited.This is the first case series that describes the re‐emergence of chronic silicosis within the tunnelling sector. As tunnelling is a major activity within large infrastructure and construction projects around Australia, this case series suggests that there may be a larger cohort of tunnellers with chronic silicosis who have yet to be diagnosed with silicosis due to their current asymptomatic states and the small numbers of persons trained to diagnose this condition, both clinically and radiologically . With alWe believe that an effective medical surveillance programme was able to be implemented in Queensland due to the increased awareness amongst employers, medical professionals, and radiologists who were qualified in ILO B reading. This increased awareness was a direct result of the cases of acute silicosis and coal workers pneumoconiosis which is unique to Queensland , 7, 8. INoting the limitations of a case series, possible hypotheses for the re‐emergence of chronic silicosis include the increased number of construction projects in areas with high silica content. Other hypotheses include poor occupational hygiene practices – such as suboptimal ventilation and over‐reliance on ineffective personal protective equipment. A further hypothesis could be that the published very low incidence rates resulted in a decreased perception of risk. This faulty perception could have led to a reduced focus and adherence on traditional occupational hygiene practices that have been demonstrated to be effective.The medical surveillance programme that was undertaken looked at all workers who were employed at tunnelling sites. As such, the cohort from which our case series was drawn comprised of administrative personnel, senior and middle management, and ancillary site staff in addition to those persons who worked underground. We postulate that there may be a prevalence of up to 10% if the tunnellers were categorized into a homogenous exposure group.Due to the specialized nature of tunnelling, tunnellers frequently move across various construction projects in Australia and are often employed by several different companies during their working career. Silicosis screening programmes are currently state‐based and therefore this cohort of workers are at this potential risk of being missed as they move across state boundaries. In addition, the state‐based nature of workers compensation schemes does not easily accommodate workers who have acquired silicosis across multiple jurisdictions.This case series also identifies a need for improved dust monitoring programmes. This need is linked to not only preventing the causation of the occupational lung disease, but also in assisting fitness for work determinations and improving work‐related outcomes for these individuals. There will also need to be greater attention paid to engineering controls and personal protective equipment within these underground work environments.In conclusion, the re‐identification of black lung and silicosis in the mining industry in Queensland in 2015 has led to a greater awareness of silicosis in other industries. In Queensland, screening for silicosis has been expanded beyond the coal industry to artificial stone workers, quarry workers, and hard rock miners. This cluster of eight cases in tunnellers supports the need for further expansion of screening in construction and infrastructure workers, particularly those in high‐risk roles such as those involved in underground tunnel projects. The re‐emergence of chronic silicosis will pose new and different healthcare challenges within this larger industry which is potentially fragmented in its regulation. It is envisaged that the increase in the chronic silicosis burden of disease will add to the growing public health interventions around pneumoconiosis over the coming years.Appropriate written informed consent was obtained for publication of this case series and accompanying images.


Orthotopic bladder substitution (OBS) is a management option for urinary diversion in men and women undergoing cystectomy. The aim of the procedure is to provide a functional continent urinary reservoir of adequate capacity, compliance and low pressure. We have provided a narrative review of the existing literature and highlighted areas where improvement and standardization can be recommended.Literature search included database search for publications from January 1970 to November 2020, using keywords including OBS, bladder reconstruction, neobladder, radical cystectomy, robotic cystectomy, intracorporeal neobladder, surgical technique, patient selection and outcomes.Due to various factors including indications, operative technique and risk of complications, OBS is an enormous undertaking and commitment for patients, surgeons and health professionals involved in the care pathway. The main considerations for patient selection, the technical elements of the procedure and the rationale behind these are discussed. Previously considered to be a choice for a select few, the inclusion criteria have expanded over the last decade. Similarly, surgical techniques including the choice and configuration of bowel segments, construction of anastomosis and nerve or organ sparing procedures have evolved over the years. Minimally invasive laparoscopic and robotic assisted surgery has added further perspectives to the existing literature on OBS. Understanding the principles of operative techniques and assessing the best evidence to influence patient management is crucial as it has a major impact on clinical outcomes. Peri‐ and post‐operative care, focused on the prevention of complications and morbidity, affects long‐term functional and oncological outcomes, which ultimately dictates the quality of life.This concise overview of OBS literature highlights the importance of pre‐operative, peri‐operative, and post‐operative aspects with regards to the optimization of patient care. To achieve the best results, meticulous attention should be paid in all these areas, surgical and multi‐disciplinary. Patient education and counseling, with shared decision making are central to the success of the procedure. FSfrozen sectionISCintermittent self‐catheterizationLOSlength of stayOBSorthotopic bladder substitutionONBorthotopic neobladderQoLquality of lifeRCTrandomized control trialRCradical cystectomyRPradical prostatectomyUDurinary diversionWHOWorld Health Organization1The term orthotopic refers to a bladder substitute (neobladder) reconstructed in the same place as the native bladder, that is, in the pelvis. It is anastomosed to the native urethra, with a functional external urethra sphincter providing the continence mechanism. Orthotopic bladder substitution (OBS) was first described for male patients after radical cystectomy for cancer in 1913 by Lemoine using rectum.In a consensus statement by the World Health Organization (WHO) and the Société Internationale d’Urologie (SIU), neobladder reconstruction was reported in 47% of patients undergoing radical cystectomy and urinary diversion.Patients requiring RC are often referred from the center of initial diagnosis to a tertiary center for oncological management. An understanding of pre‐operative considerations, operative techniques, and post‐operative care is essential for the optimization of patients undergoing neobladder reconstruction. We outline the fundamentals of OBS primarily in the context of radical cystectomy. The essential steps to prevent pitfalls from the beginning of the patient journey and to achieve long‐term successful outcomes are discussed.2A Medline database search using the following keyword search criteria was performed: OBS, bladder reconstruction, neobladder, radical cystectomy, robotic cystectomy, intracorporeal neobladder, surgical technique, patient selection, and outcomes. All publications from January 1970 to November 2020 were included and the literature search was confined to publications in English. An additional manual search of references in relevant published articles was performed.3Bladder substitution with intestinal segments is an option for continent diversion performed after cystectomy. The 2nd International Consultation on Bladder Cancer recommendations on the reconstructive options after RC (2012), have outlined criteria for patient selection with regards to renal function and risk of secondary tumor.3.1For OBS following radical cystectomy, oncological aspects such as a complete resection of the primary tumor, negative margins, and the absence of metastatic disease are important. Pre‐operative transurethral biopsies from the prostatic urethra in males and bladder neck in females have a high negative predictive value, although studies show that they are not as accurate as intra‐operative frozen sections.3.2Patient profile is of utmost importance and careful consideration of co‐morbidities, performance status, life‐expectancy, and cognitive ability is needed. There is no age limit at which OBS can be offered, and it has been suggested that elderly fit patients have similar outcomes to their younger counterparts.3.3Functional abnormalities of lower urinary tract including urethral strictures should be excluded. External urethral sphincter dysfunction and stress urinary incontinence are relative contraindications. Pelvic irradiation is not considered an absolute contraindication; however, continence rates are lower (56%‐76%).3.4All options for urinary diversion including ileal conduit should be discussed with the patient and shared decision‐making should be implemented. Involvement of a specialist nurse early on in the management pathway is essential. Initiation of the Enhanced Recovery Protocol for RC has shown to reduce median LOS from 14 days to 7 days in the UK4The main principle is to provide a continent urinary reservoir of adequate capacity, compliance, and low pressure. The advantages are continence (and the avoidance of a stoma bag), potential for spontaneous voiding and improved body image.Careful dissection around the urethral margin to preserve the optimal length of urethra is necessary. Intra‐operative frozen section is recommended to confirm negative margins. Prostatic urethral tumors are associated with an increased risk of secondary urethral tumors (12%‐18%),4.1Ileum is the most commonly used bowel segment as it has better compliance and less contractility as compared with colon or cecum. Ileal neobladder can obtain the same capacity as a colonic neobladder, but ileal storage pressures are lower,4.2The chosen bowel segment is detubularized and the segments are reconfigured to create a spherical‐shaped reservoir. This obtains the maximal volume from the minimum absorptive surface area of bowel. Detubularization also lowers the reservoir pressure, reducing the risk of vesicoureteric reflux, and lowers the risk of spontaneous contractions causing urinary leak. However, it also increases the risk of requiring intermittent self‐catheterization (ISC). Examples of ileal neobladder include the Studer, Hautmann, Vescica ileale Padovana (VIP), Orthotopic Kock pouch (or hemi‐Kock), and Camey II neobladder. Table 4.3Anastomosis between the reconstructed bladder outlet and the urethra (preserving the sphincter) is created in a manner that it sits flat in the pelvis. A funnel‐shaped outlet is not favored as it has risk of kinking and mechanical obstruction. This is especially important in women as urethral‐pouch flexion can lead to chronic retention.4.4The question of whether reflux in a low‐pressure reservoir such as OBS is clinically important has been debated. The WHO Consensus Group reported that reflux is not of clinical importance in OBS, but an anti‐reflux mechanism is essential in continent cutaneous pouches.4.5Nerve‐sparing in men is performed using the same technique as radical prostatectomy by dissection of the plane between the prostatic capsule and the neurovascular bundle. Nerve sparing can be unilateral on the non‐tumor bearing side so that oncological safety is not compromised. In both male and female patients, preservation of autonomic innervation has a positive impact on continence with long term benefits.4.6With the advent of minimally invasive and robotic‐assisted surgery, techniques for ONB reconstruction have also shown advancement, though the basic principles remain the same. Robotic cystectomy and extracorporeal neobladder formation involve the construction of the ileal pouch in an open manner followed by urethro‐enteric anastomosis performed with the robot using the “Van Velthoven technique”.Figure 5In the post‐operative period, management of catheters is important, with regular flushing and removal of mucus and blood clots from the neobladder. Removal of ureteric and suprapubic catheter depends upon institutional practice and there is no consensus on the placement or duration of catheters. As per Studer's original description,6The aim of follow‐up is the early detection of metabolic complications, management of voiding and sexual dysfunction and surveillance for cancer recurrence. There are no specific guidelines for post‐OBS follow‐up, and most regimes are guided by radical cystectomy follow‐up protocols as per the published EAU guidelines.Late complications , at a mean follow‐up of 48 months, daytime and nocturnal continence rates were 99% and 78%, respectively.The 2012 Cochrane review of urinary diversion and bladder reconstruction identified studies indicating 0%‐70% of patients who require ISC, while the remainder are able to empty the neobladder by abdominal straining.Rates of erectile dysfunction in men without nerve‐sparing have been reported to be around 35% at 12 months follow‐up and are higher than that for ileal conduit (9.8%).6.2From an oncological perspective, there is no specific schedule recommended for follow‐up due to the limited data on surveillance.7Overall QoL remains good in most patients with OBS, with few differences between different types of diversion.A few studies have shown an increased benefit in QoL specifically with OBS,At medium‐term (5‐year) follow‐up (mean 89 months), general health QoL was generally well maintained as compared to the “normal” population, unless patients required ISC or suffered from daytime incontinence.Several studies have assessed and compared the quality of life in patients undergoing different types of urinary diversion. Validated questionnaires such as EORTC QLQ‐30C, SF‐36, and FACT(‐BL) (‐G) (‐VCI) do not necessarily focus on the type of urinary diversion or specific functional outcomes and sexual function. Health‐related quality of life (HRQoL) was previously reported to be only marginally better with OBS compared to Ileal conduit diversion.8OBS is a valuable surgical option with the possibility of restoring the lower urinary tract function to achieve continence. This is important for patients undergoing RC as the “loss of an organ” is somewhat compensated by the psychological benefit of a “new one.” Over the years, indications for OBS have been refined and if strategically organized, the interplay between operative techniques and post‐operative care can lead to the most successful results. Further research is required in areas of minimally invasive techniques and standardization of long‐term management. Patients should be appropriately counseled regarding the long‐term outcomes and impact on the quality of life. Above all, the type of urinary diversion should be acceptable to the patient whose main concern is cancer recurrence.None.


Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease. Traffic of dietary cholesterol from the endocytic pathway to the ER is an essential step in cholesterol homeostasis but the mechanism of this cholesterol transport is not completely understood. Sites of contact between the membranes of endocytic and non‐endocytic organelles are emerging as important regulators of both vesicular and non‐vesicular cholesterol transport. Here, we review current understanding of these membrane contact sites and their importance in redistributing dietary cholesterol from endocytic organelles to other cellular membranes and roles in neurodegenerative disease. ERendoplasmic reticulumLElate endosomesLyslysosomesCholesterol is an essential constituent of cell membranes, maintaining membrane integrity and limiting permeability, but its distribution between different cellular membranes varies considerably. Cholesterol is particularly enriched at the plasma membrane . LDL is comprised of a cholesterol ester/triglyceride core surrounded by a phospholipid and unesterified (“free”) cholesterol shell, with a protein component (apoB‐100) that acts as a specific ligand for the LDL receptor (LDLR). LDL enters the endocytic pathway through clathrin‐mediated LDLR endocytosis. Progressive hydrolysis by acid lipases releases free cholesterol that is delivered by the small luminal protein Niemann Pick type‐C protein 2 (NPC2) to the large transmembrane protein NPC1 on the endo/lysosomal limiting membrane.Cholesterol sensing in the ER by Insig retains SCAP‐SREBP complexes in the ER. When cholesterol transport to the ER is low, however, SCAP‐SREBP is transported to the Golgi. Proteolytic cleavage by Golgi‐resident proteases releases a soluble SREBP fragment that relocalizes to the nucleus to upregulate transcription of cholesterol biosynthesis and uptake genes.Delivery of dietary cholesterol to target cellular membranes involves a complex relationship between both vesicular and non‐vesicular transport mechanisms. The role of vesicular traffic in delivering material from one organelle to another has been widely studied, but only recently have we begun to appreciate the importance of non‐vesicular transport in inter‐organellar communication. Non‐vesicular lipid transport is mediated by membrane contact sites (MCS), regions of close membrane apposition (5‐30 nm) between neighbouring organelles. MCS are stabilized by tethering complexes that maintain close proximity between the opposing membranes, but without membrane fusion. These tethers can often be discerned by electron microscopy, as multiple strands between the opposing membranes of the two organelles.Since their identification only a decade ago,11.1The most widely studied MCS population formed by endocytic organelles is the ER‐endosome contact. The ER forms extensive MCS with endocytic organelles, which increase during endosome maturation, with approximately 90% of lysosomes estimated to form an ER contact site at any given time , through which it can interact with the MSP domain of ER‐localized tethering proteins VAPUnlike STARD3, which is anchored to endosomal membranes by its MENTAL domain, ORP1L is recruited to LE by the small GTPase, Rab7, which plays a number of key roles in late endocytic membrane traffic. The extended contact between Rab7‐positive LE/Lys compared with earlier (Rab7‐negative) endosomes1.2Endocytic organelles have been shown to make contact with most organelles in the cell, including the Golgi, peroxisomes and mitochondria. While much less is known about the architecture of these MCS, progress has been made and several proteins with roles in contact site formation or function (or both) have been identified. Of these, the MCS between endocytic organelles and Golgi are among the better defined. Endosome/lysosome‐Golgi MCS were first identified in response to nutrient sensing. Under conditions of starvation, a tumour suppressor protein, folliculin, associates with lysosomes to drive lysosome MCS with Rab34‐positive Golgi membranes through direct interaction with the Rab34 effector RILP, with a consequent retention of lysosomes in the peri‐nuclear compartment.Mitochondria crosstalk with the endocytic pathway, especially through the activity of AMPK, is central to the coordination of mitochondrial and lysosomal function22.12,The majority of LDL‐derived cholesterol is reported to be trafficked to the PM, to safeguard supply, before onward transport of excess cholesterol to the ER for esterification and storage in lipid droplets.3P on LE. At these contacts, the microtubule motor protein kinesin‐1 is transferred from protrudin to the late endosomal motor adaptor FYCO1 to mediate plus end directed transport of LE to the plasma membrane. In contrast to the Rab8‐dependent pathway, however, these endosomes undergo synaptotagminVII‐dependent fusion with the plasma membrane to support membrane protrusion and neurite outgrowth. Although the impact of this pathway on cholesterol distribution has not yet been explored, it likely offers a mechanism of sterol delivery from the limiting membrane of LE to the plasma membrane.Another potential pathway for delivery of LDL‐derived cholesterol to the plasma membrane culminates in endosome‐plasma membrane fusion. ER‐localized protrudin tethers ER‐endosome MCS through coincident binding of Rab7 and PIThe endocytic recycling compartment is estimated to contain approximately 30% of total cellular cholesterol, the majority of which is delivered to the plasma membrane in an ATP‐independent manner, indicative of non‐vesicular transport.2.2Several lines of evidence point to the Golgi as an intermediate on the endosome to ER cholesterol transport pathway. Depletion of SNARE proteins involved in vesicular traffic between the endosome and the TGNThe finding in 2014, that MCS define the position and timing of endosomal fission,In addition to vesicular retrograde traffic, a non‐vesicular transport mechanism from Rab11‐positive recycling endosomes to the TGN at endosome‐Golgi contacts has recently been reported. Work from Sobajima et al identified a Rab11‐GTP and OSBP binding protein, RELCH that tethers recycling endosomes to the TGN to mediate cholesterol transport.2.3Mitochondria have the lowest abundance of sterols among cellular organelles, approximately four and 40 times less than ER and plasma membrane, respectively.In yeast, mitochondria form MCS with the vacuole (yeast lysosome), called vacuole and mitochondria patches (vCLAMPs), which are regulated by a complex consisting of the Rab7 GTPase‐like Ypt7 and one of its effectors Vps39/Vam6 subunits of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex and the mitochondrial protein Tom40.NPC1 or NPC2 genes. NPC2 protein mobilizes LDL‐derived cholesterol in the lysosomal lumen and subsequently transfers it to NPC1 for egress from the limiting membrane. Depletion of NPC2 in CHO cells expressing the CYP11A1 F2‐protein, reduced pregnenolone formation to the same extent as STARD3 depletion, but combined STARD3 and NPC2 knockdown had no additional effect on pregnenolone synthesis.In mammalian cells, early evidence for a complex able to transfer cholesterol to mitochondria came from studies on STARD3. STARD3 is anchored to the cytosolic side of late endosome through an MLN64 N‐terminal (MENTAL) membrane domain highly homologous to another transmembrane protein, STARD3NL. STARD3 also possesses a START sterol binding domain at the C‐terminal. Overexpression of STARD3 in steroidogenic competent cells modestly increases pregnenolone formation and expressing STARD3‐START domain alone strongly increases steroid formation.2.44,5P2 on the peroxisome membrane to tether connections between the two organelles.Like lysosomes, peroxisomes are organelles bound by a single membrane that participate in metabolic pathways. Under conditions of high LDL‐cholesterol in the endocytic pathway, lysosomal Synaptotagmin VII binds PI2.52 and PIP2 increase the rate at which ORP1L extracts cholesterol from the membrane to regulate egress of cholesterol from LE/Lys. This could underlie the inhibition of mTORC1 by PIP2 accumulation on LE/Lys. LE/Lys cholesterol can activate mTORC through SCL38A9,2 enhancement of ORL1L‐mediated cholesterol egress could result in mTORC1 inhibition.While the majority of cholesterol transport between the endocytic pathway and the ER is believed to occur via the plasma membrane, it is estimated that direct transport mechanisms between LE/Lys and the ER account for approximately 30% of LDL‐derived cholesterol delivery to the ER.NPC1 has an established role in cholesterol egress from late endocytic organelles and although the handover of cholesterol from NPC2 on the lumen of LE/Lys to NPC1 on the membrane, has been structurally defined,The role for NPC1 in MCS regulation coupled with the finding that MCS expansion can restore cholesterol transport in NPC1‐deficient cells, underscores the importance of MCS in cholesterol egress and further points to the existence of an alternative, NPC1‐independent mechanism, for cholesterol passage across the glycocalyx. NPC2 has been proposed to mediate cholesterol transfer directly to the limiting membrane, as well as to other transmembrane proteins.As a key role of MCS in cholesterol egress continues to emerge, so too does an association between defects in MCS proteins and neurodegenerative disease Table . The mecIn summary, we are beginning to appreciate the important role played by MCS in cholesterol homeostasis and the complex network of organelle interactions involved. By studying rare neurodegenerative diseases associated with defects in MCS formation, much is being learnt about inter‐organelle sterol trafficking. These advances are paving the way for innovative new treatment strategies.The authors confirm they have no conflicts of interest to report.