The upregulation of anti-inflammatory genes was observed in Trem2hi macrophages, and an in vivo injection of soluble Trem2 during the subacute phase of MI significantly improved myocardial function and the remodeling of infarcted mice hearts, suggesting the potential therapeutic role of Trem2 in LV remodeling. Further investigation of the reparative role of Trem2 in LV remodeling would provide novel therapeutic targets for MI.Advances in interventions after myocardial infarction (MI) have dramatically increased survival, but MI remains the leading cause of heart failure due to maladaptive ventricular remodeling following ischemic damage. Inflammation is crucial in both the initial response to ischemia and subsequent wound healing in the myocardium. To date, preclinical and clinical efforts have been made to elucidate the deleterious effects of immune cells contributing to ventricular remodeling and to identify therapeutic molecular targets. The conventional concept classifies macrophages or monocytes into dichotomous populations, while recent studies support their diverse subpopulations and spatiotemporal dynamicity. The single-cell and spatial transcriptomic landscapes of macrophages in infarcted hearts successfully revealed the heterogeneity of cell types and their subpopulations post-MI. Among them, subsets of Trem2 Survival and the clinical outcomes of acute myocardial infarction (MI) have improved over the years, owing to major advances in cardiac revascularization strategies and the introduction of various pharmaceutical interventions . HoweverAfter myocardial infarction, ventricular remodeling or recurrent ischemic events are both reported to be associated with inflammation . In partSpecifically, macrophages have been suggested to play a pivotal role in the clearance of infarcted tissues, as well as in wound repair and remodeling processes . Subsetshi macrophages and the soluble form of triggering receptor expressed on myeloid cells 2 (Trem2), dominant anti-inflammatory immune cells in infarcted myocardium and its key molecule, leading to significant functional and structural improvements in infarcted hearts.In this article, we review major studies to answer these long-standing questions to investigate these underlying inflammatory processes and the spatiotemporal dynamics of MI-associated immune cells. In particular, we focus on the role of Trem2LV remodeling is a process in which the heart adapts to mechanical, neurohormonal, and inherited changes by regulating the size, shape, and function of the ventricle. It is commonly observed in infarcted hearts . AdverseInflammation has been suggested to be a classic pathogenesis that plays a key role in atherosclerosis, both in the early phase of initiation and the long-term progression of vulnerable plaques . The JUPA number of clinical studies have been conducted on MI models to identify inflammatory molecules or responsible immune cells and, ultimately, to find novel therapeutic targets. The CANTOS trial demonstrated not only that increased serum levels of high-sensitivity C-reactive protein (CRP) and interleukin (IL)-6 were associated with elevated cardiovascular risks but also that the blockade of IL-1 beta with a monoclonal antibody in patients with a history of MI and an elevated baseline level of CRP was followed by significantly lowered all-cause and cardiovascular mortalities . This st+ cardiac-resident macrophages [+ monocytes after MI also reflect the mobilization of monocytes from bone marrow and recruitment [hi CD16-) are associated with several diseases hallmarked by inflammation and play an important role in the initiation and progression of the inflammatory response [low CD16hi) monocytes, in contrast, express high levels of CX3CR1 and low levels of chemokine receptors, including CCR2- and Ly6Clow, and they play a role in the resolution of inflammation [Resident cardiac macrophages play a housekeeping role in cardiomyocytes, such as promoting coronary artery development or maintaining tissue homeostasis . Macrophrophages . Interacrophages . An acutrophages . The charuitment . Human mresponse . Nonclasammation .hi macrophages dependent on CCR2/CCL2 signaling in the earlier phase and regenerative Ly-6Clow macrophages via CX3CR1 signaling in the later phase. Classically, macrophages, defined as the M1 phenotype, infiltrate on days 1 to 3 after MI and promote the phagocytosis of necrotic cells by activating inflammation, while those with the M2 phenotype on days 5–7 after MI are responsible for the reconstitution of infarcted tissue and the resolution of inflammation  were upregulated in Ly-6Chi monocytes and early macrophages, while their expressions gradually decreased to low levels in late macrophage populations  were downregulated in Ly-6Chi monocytes and early macrophages but gradually upregulated in late macrophages during the course of MI. In particular, Trem2 was particularly upregulated in late-macrophage subsets rather than in early subsets  10 and DAP12. Trem2 binds to various molecules and interacts with other signaling pathways in complicated manners. Its downstream signaling may represent a danger signal in tissue damage or disease, while it is known to have a physiological role in tissue development or maintenance in specific microenvironments, such as those of the brain or bone. Its biological role is known to significantly modify cellular functions via the induction of phagocytosis, the restriction of inflammation, and the promotion of macrophage survival ,47.+ CD9+ macrophages in human fibrotic livers [Previous studies investigating the association between Trem2 and heterogenous diseases, including cardiovascular disease, are depicted in c livers .hi macrophages increase lipid metabolism in atherosclerotic plaque. Moreover, a high expression of Trem2 can reduce the stability of plaque. In Trem2hi macrophages, researchers observed increased levels of cathepsins, which could increase plaque vulnerability and the inflammation of atherosclerosis [In cardiovascular disease, recent studies support the role of Trem2 in atherosclerosis. Atherosclerotic plaque is formed by accumulating fat-rich macrophages due to the recruitment of circulating monocytes and the differentiation and proliferation of local macrophages . Recentlclerosis . Trem2 mclerosis ,57.hi macrophages in the mouse heart during the progression of atherosclerosis, which did not align with the M1 or M2 profile [hi macrophages infiltrate specific areas or time points post-MI using concomitant analyses [hi macrophages. They orthogonally validated the expression of Trem2 in the heart after MI through immunohistochemistry, Western blotting, co-localization assays, flow cytometry, and quantitative RT–PCR  beginning on day 3 post-MI and peaking on day 7. They injected sTrem2 mixed with a gelatin hydrogel (sTrem2-GH) in vivo and identified that sTrem2 promotes functional and structural improvements in damaged hearts in mice (Monocytes and macrophages are known to play key roles in ventricular remodeling after MI. Nevertheless, the effectiveness of targeted immunotherapy strategies needs to be improved, and strategies sharing specific targets show conflicting results. Many recent preclinical studies have also focused on modifying monocyte responses or targeting the CCL2/CCR2 axis to suppress monocyte-trained immunomodulators after MI inflammation ,22. Appl in mice . At 28 dhi macrophages as a possible modulator in cardiac remodeling after MI, further studies are needed to evaluate their systematic properties before conducting human clinical trials. The origin of Trem2hi macrophages, sets of cells affected by sTrem2, and interacting intracellular signaling cascades should be elucidated to allow for a better understanding of their regenerative effects in infarcted hearts. Additionally, the molecular interactions and ligands of Trem2 in human macrophages need to be compared and evaluated before targeting Trem2hi macrophages in humans. Furthermore, potential side effects should be considered, such as impaired immune responses, unintended off-target effects on other signaling pathways or cellular functions, and potential neurodegenerative effects, as Trem2 is also expressed in the central nervous system and is involved in microglial function regulation.Despite the identification of Trem2hi macrophages show promise due to their protective inflammatory properties. In an in vivo experiment with mice, a soluble Trem2 injection during the subacute phase of MI significantly improved myocardial function. Further investigation into the role of Trem2hi macrophages in the post-MI immune response and the regulatory signaling of Trem2 will aid in the development of novel therapeutic strategies for MI.The spatiotemporal dynamics of immune cells during the acute phase of post-MI have recently been studied, shedding light on their complex interplay. This new understanding shifts the paradigm of myocardial cellular composition after MI and provides potential targets for MI treatment. Among the heterogeneous macrophage populations, Trem2


The field of epithelial stem cell development has been irrevocably shaped by the work of American scientist Howard Green, whose breakthroughs in stem cell culture methods translated to therapeutic practice. In this review, we chronicle the milestones that propelled the field of regenerative medicine of the skin forward over the last fifty years. We detail the early discoveries made by Green and his collaborators, highlight clinical cases that made life-saving use of his findings, and discuss the accomplishments of other scientists who later innovated upon his discoveries. Epithelial tissues such as the skin and digestive tract line the outer surfaces and inner cavities of organs, and are comprised of multilayered sheets or a monolayer of cells, respectively, that are held together primarily by E-cadherin-mediated adhesion forces . EpithelThe epidermis is a classic example of epithelial tissue and acts as a barrier against the external environment. Tight junctions, a network of particle strands mainly comprised of transmembrane proteins, form a paracellular barrier against epidermal dehydration. This function is mediated by adhesion molecules, of which claudin-1 is the most prominent . CorneocEpidermal components exhibit immune competence against pathogens through Langerhans cells and tissue-resident macrophages that function as antigen-presenting cells, as well as through keratinocyte secretion of cytokines and chemokines to initiate inflammatory responses . Within Epithelial homeostasis is maintained through self-renewal of the basal layer of the epidermis; detachment of proliferating cells from the basal layer precedes terminal differentiation and eventual detachment from the outer surface of the skin . EpithelOur current understanding of epithelial stem cell behavior can be traced through functional milestones: advancements in culture techniques, examination of epithelial cells and tissues under homeostatic and tumorigenic conditions, developmental studies to pinpoint cell population origin, and clinical breakthroughs to improve patient care. In this review, we highlight and follow the impact of those milestones on the field.The progressive advancement of epithelial stem cell research  cannot bTodaro and Green created the original 3T3-Swiss albino cell line (referred to at the time as 3T3) in 1962 from primary cultures of Swiss mouse embryonic fibroblasts. Their paper detailed the lengthy process of the primary cell culture, which first required affirmation of the correlation between growth rate and inoculation size. The optimal parameters for cell plating density were also established: too few cells resulted in zero population growth, while too many caused cell crowding and impeded growth by contact inhibition. By the 15 to 30 generation mark, the growth rate was consistent and remained so when those parameters were followed.Upon reaching this milestone, Todaro and Green declared those cultures to be established lines with significant replating viability and cultured them past a hundred generations with no signs of replicative senescence. The pair concluded that high inoculation size favors cell line establishment, and that the threshold at which cell growth ceases is higher in cells that were continually cultured at high densities than in those continually cultured at low densities, as was the case for the 3T3 lines . The oriIn the twelve years between the 1963 paper and further developments with the 3T3 lines, various additional insights in epithelial tissue development were made beyond the epidermis. Improved understanding of the thymus was underway, with attempts made to determine the origin of thymocytes: lymphoid cells present in the thymus that undergo transformation into T cells. At the time, the debate over this origin comprised contradictory data . One sidRheinwald and Green published two papers in 1975 that established the potential of the 3T3 cell lines in feeder technology. One of their works discussed establishment of a keratinizing cell line that was derived from a mouse teratoma. At the time, the term teratoma was considered imprecise because it made no assertions about structure or behavior; the notion of subdividing the word for better specificity according to malignant behavior remained in question. The general consensus, however, was thus: a teratoma was a real tumor with malformed development, grew rapidly, almost always contained malignant elements comprised of stem cells , rarely metastasized, was highly transplantable, and usually killed the host by causing emaciation . The modThe clone that would become Rheinwald and Green’s keratinizing cell line was derived from a solid tumor grown by Leroy Stevens, created through the graft of a mouse embryo onto the testis of an adult mouse that was given to Rheinwald and Green . The tumThe second paper published by Rheinwald and Green focused on the keratinization of non-malignant human diploid epidermal cells . OptimizOne intriguing note from both 1975 papers ,29 is thThis epithelial cell stimulation by hydrocortisone was not restricted to epidermal keratinocytes. In 1978, Gaffney and Pigott detailed hydrocortisone use in human mammary epithelial cell culture and found that its pairing with fetal bovine serum  increased cell cluster attachment and colony formation by 2.5-fold . FBS andThe science of grafting and transplant remains a continual balance between technological advancements and the limitations of the human body. To push those limitations requires innovation, and such was the case with the concept of an autograft: tissue surgically transferred from healthy flesh to a wound on the same individual. The autograft dates back in Western consciousness to surgeon Jaques-Louis Reverdin ; while hThough monumental, these achievements centered on extant tissues for skin grafts—whether taken from elsewhere on a patient’s own body or from cadavers. Problems arose when burns were suffered to such a degree that there were simply not enough skin grafts available to cover swaths of wounds. The 1975 Rheinwald and Green papers described above ,29 laid In 1979, Green and collaborators Kehinde and Thomas explored this possibility. Green’s cell medium was further optimized through the additions of epidermal growth factor (EGF) and cholera toxin; the former increased culture growth rate and colony-forming efficiency while the latter increased the levels of cellular cyclic AMP (cAMP). The protease dispase was applied to dissociate confluent epithelia from a petri dish in a monolayer, which allowed its placement upon a transplantable collagen surface. This surface was a clear matrix  made of In 1984, this technology was applied to life-saving effect. Two children, each with burns covering over 97% percent of their body area, were biopsied for full-thickness skin samples that were minced to create single cell suspensions. From these, cell sheets between two and eight cell layers thick were grown and lifted with dispase. These cultured epithelial autografts were placed onto gauze and applied directly to the wounds. Within two to three weeks, the epithelium achieved uniform thickness. Development of normal epidermal structures such as the horny and granular layers was confirmed. By 21 to 23 weeks, both patients’ wounds were over 85% healed with roughly half of epithelial coverage comprised of cultured epithelial autografts .This breakthrough reached a stumbling block by the 1990s: burn units reported consistently poor clinical outcomes with this technique for massive full-thickness burns. Graziella Pellegrini theorized in 1999 that these poor outcomes were owed to the loss of holoclones—a term and concept first coined by Barrandon and Green . A holocTo maintain the integrity of cultured autografts and increase the potential for post-transplant epidermal regeneration, Pellegrini and collaborators used fibrin to cultivate keratinocytes: fibrin being a natural substrate already abundant in the wound healing bed . The estPellegrini’s study was comprised of seven patients from infancy to 30 years of age who suffered full thickness burns on 20% to 45% of their bodies. Fibrin gels were prepared from fibrinogen and thrombin solutions , thrombiBeyond holoclone loss and autograft detachment woes, poor clinical outcomes reported from burn units were attributed to the lack of a dermal component. Several approaches were developed to combat this problem, one being the application of cryopreserved dermal allografts from cadavers onto the burn wound surface. The immunosuppression caused by thermal damage paired with the lesser immunoreactivity of the dermis (as compared to the epidermis) allowed allograft persistence. The epidermis formed from the allograft was stripped off a month after transplant and replaced by cultured epidermal autografts .Another approach was artificial skin, which faced the twofold task of early wound closure followed by long-range function and biodegradable membrane disposal. In 1979, Silver, Yannas, and Salzman crosslinked bovine hide collagen with the glycosaminoglycan (GAG) chondroitin 6-sulfate, which decreased the pro-coagulant and the platelet aggregation activity of the collagen and allowed its potential use as a blood-compatible vascular replacement material in vitro . Yannas In 1981, Yannas, Burke, and collaborators published on the use of artificial skin, comprised of a collagen-chondroitin 6-sulfate fibrillar dermis and a Silastic (silicone elastomer) epidermis, on ten third-degree burn patients aged from 3 to 60. The human type IV collagen of the dermis was derived from cadaver kidneys. The collagen-GAG dermis was applied to support host cell and vessel ingrowth in patients, while the Silastic epidermal portion was temporary at initial treatment and later replaced with autografts. The extensive wounds on all patients were successfully closed by this method with no infections or inflammatory responses .In a 2000 paper, Horch and collaborators acknowledged the progress that fibrin represented and sought to avoid the physical stress that dispase caused to autografts during cellular release from culture surfaces. Because dermal equivalents did not have blood vessels for transport and might block nutrients from reaching the keratinocytes atop them, Horch detailed an “upside-down” method in which subconfluent human keratinocyte monolayers were put in direct contact with fresh wounds on nude mice such that the collagen faced the external environment.2), ease and simplification of graft handling procedures, and the preservation of cell adhesion through the lack of enzymatic detachment by the “upside-down” method [By day 14, the wounds had decreased in size. By day 21, reepithelialization had taken place; well-vascularized tissue was present beneath the epidermis that stained positive for anti-human HLA, as opposed to negative-staining granulation tissue in the mice that were not given keratinocytes. Horch demonstrated the growth of human keratinocytes on type I collagen without a feeder layer, the minimum amount of keratinocytes needed to reconstitute an epidermis in 14 days , the former developed smaller colonies than the latter and showed a smaller percentage of Ki67-positive cells at later stages .As well as the effect of p63 on thymic epithelial stem/progenitor cells, our paper tested the role of p63 in epidermal stem cells. p63-positive rat epidermal cells generated holoclones, meroclones, and paraclones when grown in clonogenic culture that were similar to the patterns of human epidermal cells in vitro, which enabled their use as a model. Transduction with the lentivirus that expressed shRNA against p63 transcripts showed a 90% reduction of p63 protein levels in these epidermal cells by Western blot, compared to cells transduced with a control shRNA lentivirus. By days 10 and 14, the stem cells with an shRNA-induced reduction of p63 formed far smaller colonies than control cells. Our lab concluded that while p63 is not required for stem cell commitment and differentiation during tissue morphogenesis, it is indispensable for maintenance of the proliferative potential of epithelial stem cells . This knNanog was implicated in this process, it was ultimately found disposable for iPS cell induction and maintenance [To discuss the final component of Green’s epidermal keratinocyte propagation technique, we must first mention two new individuals: Kazutoshi Takahashi and Shinya Yamanaka. Through an exhaustive process of elimination, it was determined that the transcription factors Oct3/4, Sox2, c-Myc, and Klf4 in tandem with each other are able to reprogram differentiated cells into induced pluripotent stem cells (iPS cells) that exhibit the properties of embryonic stem cells. Though a fifth gene called ntenance .As with Green’s cultured epithelial autografts, Takahashi and Yamanaka’s groundbreaking discovery came with key caveats. The viral vectors used for transfer and induction of what became known as the Yamanaka factors were found to increase tumorigenicity and cause aberrant transcription, which hampered potential clinical use ,58. JustOct4::GFP transgenic mouse embryonic fibroblasts that were transduced with Oct4, c-Myc, and Klf4. Further, 1 compound from a library of 800 with known pharmacological targets was added to each well of a 96-well plate. Three wells yielded GFP+ colonies, which indicated the growth of mouse embryonic stem cell-like colonies in the absence of Sox2. Because the 800-compound screen included the histone deacetylase inhibitor (HDAC) valproic acid in the cell culture medium, further tests were required to see if the small molecule candidates required an HDAC inhibitor to reprogram differentiated cells. From the three candidates, only the transforming growth factor-β (TGF-β) receptor 1 kinase inhibitor E-616452 was shown to form GFP+ colonies in the HDAC inhibitor’s absence. For its ability to replace both Sox2 and c-Myc (whose absence reduced tumor formation risk), E-616452 was chosen for further characterization and given the name RepSox—Replacement of Sox2 [Ichida’s paper focused on Sox2, culturing  of Sox2 .TGF-β is a superfamily of cytokines whose receptors are ubiquitous on every cell surface . It has 2+ medium, and even then senescence was evident after only ten days [The connection between RepSox and the work of Rheinwald and Green was made years in the wake of this discovery as our lab investigated the potential of TGF-β inhibition for epithelial cell culture. Before this, the only recourse to stall rapid differentiation of primary keratinocytes was with low Caten days . The efften days .The knowledge of the TGF-β ligand pathway that leads to the nuclear translocation of SMAD2/3 , the obs2+ concentration raised, the cells were able to differentiate normally at any given passage cycle up to one year, suggesting the lack of malignant transformation after RepSox expansion. Furthermore, this finding was not restricted to keratinocytes: with RepSox, newborn and 4-week-old mouse epithelial cells from the salivary gland, tongue, esophagus, bladder, thymus, and cornea all survived expansion for at least 60 days. These cultures maintained their epithelial progenitor cells, evidenced in the production of large holoclone-like clones in 3T3-J2 co-culture and in the expression of epithelial progenitor cell-associated genes  measured by quantitative RT-PCR [Our data showed that not only did RepSox stimulate proliferative capacity, it enriched p63-positive epithelial stem/progenitor cells derived from the mouse epidermis in tandem with 3T3-J2 co-culture. This allowed for long-term survival of these otherwise rapidly senescent cells. TGF-β signaling thus correlated inversely with the clonal potential of epithelial stem/progenitor cells. When RepSox was removed and the Cae RT-PCR . With thThe two pediatric burn patients saved by cultured epithelial autografts in 1984 represented a clinical and scientific breakthrough, to say nothing of their chance to live full lives. In 2015, another pediatric patient presented with a dire condition known as Junctional Epidermolysis Bullosa (JEB). As a disease class, Epidermolysis Bullosa is characterized by extreme fragility of the skin that leads to frequent blisters despite minimal injury ; epidermLAMB3 gene, one of three genes jointly responsible for coding the basement membrane protein laminin-332; the mutation of these genes characterizes JEB.Tobias Hirsch and collaborators pursued a gene therapy approach for the patient, who had complete epidermal loss on roughly ~60% of the body surface. As usual, the Green protocol began with a biopsy and expansion of primary keratinocytes from the biopsy sample; Hirsch took this a step further and transduced the primary keratinocytes from the biopsy with a retroviral vector. This vector expressed the wildtype cDNA for the The transgenic keratinocytes maintained consistent percentages of holoclones throughout the mass culture and formed epidermal graft sheets. Ultimately, the treatment team was able to cover the patient’s extant lesions with the transgenic graft sheets. The fully regenerated epidermis spread over time to restore ~80% of the body surface. At biopsies 4, 8, and 21 months after grafting, there was no sign of blistering or erosion and the epidermis had wholly normal morphology . This caThough the 3T3-J2 cell line has been a mainstay in epithelial stem cell research since its inception, recent developments have shown the feasibility of other approaches. With the use of RepSox, our lab was successful in replacing mouse 3T3-J2 feeder cells with human feeder cells  to culture human epidermal keratinocytes. This effectively ended the reliance on 3T3-J2 feeder layers, and allowed for the creation of completely autologous skin grafts with no reliance on mouse tissues . RepSox Howard Green did not work alone for any of his seminal breakthroughs. He recognized the necessity of collaboration, freely gifting the 3T3-J2 cell line for experimental work. The successors that built upon his legacy were not themselves isolated in their accomplishments, and the whole of epithelial stem cell research is thus a testament to the collective benefits of scientific openness. The progression of gene editing to treat disease, the exploration of biomaterials and engineering for artificial skin, the establishment of long-term epithelial stem/progenitor culture, and the replacement of 3T3-J2 feeders with human feeders for fully autologous skin grafts are all owed to that openness. The field will doubtless look vastly different in another fifty years. Howard Green’s legacy, however, will remain the same.


V. cholerae O1 infection annually, and infection timing was not consistently correlated with reported cases. Four in 2,340 infections resulted in symptoms, only one of which was reported through the surveillance system. These results provide new insights into cholera transmission dynamics and burden in the epicenter of the 7th cholera pandemic and provide a framework to synthesize serological and clinical surveillance data.Our understanding of cholera transmission and burden largely rely on clinic-based surveillance, which can obscure trends, bias burden estimates and limit the impact of targeted cholera-prevention measures. Serologic surveillance provides a complementary approach to monitoring infections, though the link between serologically-derived infections and medically-attended disease – shaped by immunological, behavioral, and clinical factors – remains poorly understood. We unravel this cascade in a cholera-endemic Bangladeshi community by integrating clinic-based surveillance, healthcare seeking, and longitudinal serological data through statistical modeling. We found >50% of the study population had a  Vibrio cholerae El Tor O1 continue to ravage communities lacking access to safe water and insanitation. In contrast to the few official cholera cases reported to the World Health Organization each year from South Asia, this region likely bears a significant portion of the global burden of cholera and serves as the source of genetic diversity within the 7th pandemic lineage 3. While past surveillance efforts have provided glimpses into the burden of cholera in South Asia, their scope has been limited due to non-exhaustive testing of acute watery diarrhea cases (suspected cholera), which could result from a number of other related pathogens, and a focus on only medically-attended cases 7. Viewing cholera through the lens of passive surveillance systems alone may lead to severe underestimates of the incidence and a skewed understanding of the transmission dynamics of and population immunity related to pandemic V. cholerae O1. This understanding is critical for designing tailored interventions to curb transmission and reduce the burden of disease, especially in the posited cholera epicenter of South Asia.More than half a century into the 7th cholera pandemic, V. cholerae O1 specific antibodies – and the number of cholera cases detected through clinic-based surveillance ranging from close to 1 case per infection to well over 100 10. More recent analyses from a national serosurvey in Bangladesh suggested that 1 in 5 people have at least one infection each year with V. cholerae O1, yet fewer than 1,000 cases have been reported to the WHO annually over the past decade 12. Though both serologically inferred infections and clinical cases provide important clues about the true burden of disease, each data type has its own limitations and biases, and methods to integrate both types of data and translate one derived metric to another  are lacking.Historical estimates of cholera seroincidence from South Asia have revealed a wide gap between infections – defined by an immunologic boost of V. cholerae O1 leading to a boost in antibodies, but only a fraction of these lead to clinical disease, which can partially be explained by differences in exposure routes, inoculum size, previous exposures to V. cholerae O1 and circulating strains 8. Further, upon experiencing a symptomatic V. cholerae O1 infection, only a portion of individuals will seek care at formal medical facilities based on disease severity and access to care. While diarrhea-related care seeking data for children less than five years of age are abundant, largely through the Demographic and Health Surveys (DHS) 14, such data for older children and adults are scant, thereby limiting methods for translating estimates of infection into estimates of burden of disease. Passive clinic-based surveillance is most commonly employed for tracking cholera mainly through the symptom-based classification of suspected cholera, however, laboratory confirmation is rare and the suspected cholera case definition is known to be highly non-specific 15. A recent review suggested that, on average, less than half of clinically suspected cholera cases captured by surveillance are true infections from V cholerae O1, but this proportion varied widely over time and space 16. Understanding the pathway from infections with V. cholerae O1 to confirmed and reported clinical cholera is crucial for interpreting surveillance data, making inferences about transmission dynamics and disease burden, and ultimately improving our ability to target resources in the fight against cholera.Only a fraction of infections appear as confirmed cases within a facility-based surveillance system which can be due to a suite of host and pathogen factors . Serolog11. Refining our understanding of the links between V. cholerae O1 infections in the community and medically attended clinical cholera is key to enhancing our ability to interpret data from passive clinical surveillance and serosurveys in South Asia and elsewhere.Here, we combine data from a longitudinal serologic cohort, enhanced clinical and laboratory surveillance, and a health seeking behavior survey to elucidate the pathway between exposure to these bacteria and clinical disease, and estimate the true incidence of infections and symptomatic cholera disease in an endemic sub-district of Bangladesh Through enhanced clinical surveillance from January 24, 2021 to February 13, 2022, we detected 2,176 suspected cholera cases  at the two primary sites treating diarrhea cases in the Sitakunda subdistrict, the geographic focus of our analyses . Thirty-V. cholerae O115, we performed rapid diagnostic tests on all suspected cholera cases and PCR and/or culture on a subset. After taking into account the performance of the array of diagnostic tests used , we developed a Bayesian approach to reconstruct the unobserved infection histories of each participant in our serologic cohort taking into account both antibody kinetics and measurement error , with incidence increasing by age group  infections in the community, only one ended up as a medically attended cholera case captured by the surveillance system, and if cholera confirmation was done with traditional microbiological culture, this gap would grow even wider due to imperfect test performance .The relationship between infections in the community and observed clinical cholera cases was not constant through time. We initially conceptualized that the rates of clinical cholera and infections were proportional throughout the study period in our Bayesian model of infection incidence. However, we found strong statistical support  for an alternative model where seroincidence rates were composed of two components: one proportional to clinical cholera incidence rates and the other a constant hazard throughout the period independent of observed clinical cases, which was ultimately used in our primary analyses. This model formation, which served as the basis for our primary results, suggests that even at times with no medically attended cholera cases, more than four thousand infections  occur each week in Sitakunda.V. cholerae O1 where resulting cholera disease is the exception rather than the rule. We estimated that only 4 in 2,340 infections will result in symptomatic infections and among these, only one will seek care and ultimately be captured by the surveillance system. Passive clinic-based surveillance, the lens through which cholera is typically viewed in most of the world, captures only a fraction of symptomatic cases and obscures our understanding of the dynamics and magnitude of V. cholerae O1 infections. Our results shed new light on the continuum between infections and medically attended clinical disease in endemic populations and provide robust estimates of the true burden of cholera.Through joint inference on data from enhanced clinical and laboratory surveillance, a longitudinal serologic cohort, and a healthcare seeking behavior survey, we illustrate a landscape of regular immunologic exposures to 23. Another study conducted in six sentinel sites around Bangladesh from 2010–2011, which attempted to estimate the incidence of severe cholera by use of hospital admissions data and a care-seeking survey, found that incidence rates ranged across sites from 0.3 to 4.9 per 1,000 per year and care seeking probabilities consistent with those estimated in our study 24. Unlike previous studies, we incorporated serologically-derived incidence data and accounted for diagnostic test performance in our estimation, and approximated the true burden of cholera in this endemic setting to be 0.9 symptomatic infections per 1,000 per year. Though our estimate falls within the lower range of historical estimates, we cannot reliably attribute differences to true changes in incidence given the prior use of alternate, less sensitive methods.While several investigators and decision-making bodies have attempted to estimate the true burden of symptomatic cholera, they have primarily used facility-based suspected case data, often without laboratory confirmation. Most studies have neither adjusted for health seeking behaviors, diagnostic test performance (when used) nor severity of infection, possibly resulting in biased estimates of risk and poor allocation of resources. Perhaps the most widely used estimate of cholera incidence in Bangladesh, for several policy and planning documents, was based on an extrapolation of medically-attended cholera incidence measured in 2005 in the neighboring area of Kolkata, India  V. cholerae O1 infections, those that result in symptoms and eventually care-seeking, and those that remain subclinical (11), we estimated that at least 50% of the Sitakunda population is infected with V. cholerae O1 annually, and that >99% of these infections are subclinical. Such a high-level of infection incidence has only been documented in outbreaks in previously naive populations, similar to after the initial introduction of cholera in Haiti in 2010 25, and thus has been attributed to a lack of pre-existing immunity. The high proportion of subclinical infections observed in Sitakunda, however, could represent transient infections or exposures that do not result in gut colonization or bacterial shedding and/or asymptomatic or less severe infections that result in secondary transmission. Both of these plausible explanations have implications for our understanding of endemic cholera transmission dynamics. The observed high incidence rate in Sitakunda combined with our understanding of the reproductive number of cholera  and the biannual seasonal cholera peaks 7 are consistent with the hypothesis that not all infections lead to the same magnitude and duration of protection against clinical disease. Previous modeling work implied that mild and asymptomatic infections 30 or inadequate levels of cross-protection between serotypes 31 may lead to faster immune waning, resulting in endemic areas bearing a consistently high infection incidence, but strong empirical evidence is still lacking. Despite the apparent disconnect between infection and subsequent protection, which may be explained by heterogeneity in inoculum size, immune responses and/or infection histories, our study highlights potential hypotheses and corroborates these as targets for future investigations.Our approach allowed us to estimate and partition the overall incidence of clinical . Far greV. cholerae infections leading to clinical disease have emerged over the past 30 years: the first suggesting that infections occur through exposures to V. cholerae in the aquatic environment and the second that infections are through more proximal exposures to infected individuals, often via short-term conduits like food or water 33. We found robust statistical support that infections in Sitakunda could be explained by two components, one linked to the number of cases observed at health facilities and the other a constant hazard from an unknown source. This finding may be consistent with the dual roles of frequent low-dose exposures to environmental V. cholerae (O1 and other serogroups) and seasonal exposures to higher doses of bacteria, potentially of the hyperinfectious phenotype 34, from symptomatic individuals. As the Bay of Bengal forms the entire western border of Sitakunda, frequent exposures to aquatic flora and fauna, which have been associated with V. cholerae infection, is plausible 35. A well-documented dose-response relationship exists as higher doses of V. cholerae O1 bacteria lead to more severe disease 36, and this high dose exposure pathway may be seasonal in this endemic population. The temporal clustering of symptomatic disease may further be a result of seasonal phage predation 37, serotype switching 38, or changing routes and/or manners of bacterial ingestion 39. Future detailed models exploring the forces behind these apparent different drivers of infection could ultimately improve our understanding of V. cholerae O1 dynamics.By inferring time-varying infections, our results contribute to our understanding of the different pathways through which cholera transmission occurs. Two views on the dominant routes of V. cholerae O1 transmission in endemic areas 30. This implies that the absence of cases may not mean absence of transmission, which must be taken into account when approaching elimination. After 3 years of no confirmed cases in Haiti, a new outbreak emerged in 2022 with genomic evidence linking the bacteria to the previously circulating 2010 strain in country, with cryptic transmission from subclinical infections (in the face of waning immunity and a lack of clean water sanitation) and an environmental reservoir being plausible explanations for this 40. Knowing the factors that shape this potential disconnect is likely critical for designing interventions for elimination, like vaccines and water and sanitation, assessing risk and establishing appropriate surveillance systems.Our results not only have implications for our understanding of the dynamics of this ancient disease but may provide important practical insights for the global fight against cholera and the End Cholera 2030 roadmap. Several countries have devised national cholera control plans that rely on passive clinical surveillance and have bold targets, often including elimination of the disease by 2030. Our results confirm previous modeling results that inapparent infections are key to understanding 41. Defining the immunological markers of subclinical infections, correlates of protection, and the resulting different endpoints for clinical studies, can help us further understand the mechanisms of current vaccines and lead to the development of better ones in the years to come. Further, when exposures and risk are this pervasive, our results also strengthen the argument for safe, well-managed water systems. Individuals in our serologic cohort reported using piped water more often during the high transmission season compared to other times  has been documented for other enteric pathogens, like typhoidal Salmonellae, among Bangladeshi children 44 These concordant results could provide the impetus for aligned, cross-pathogen horizontal intervention mechanisms, specifically for clean water and sanitation.Though the most widely used cholera vaccines  protect against severe disease, it is unclear how or if they protect against subclinical infection, particularly those that may lead to onward transmission er times , suggest45. We focused on vibriocidal titers given the existence of a well-characterized post-infection kinetic model from medically attended cholera cases calibrated on mostly adults 22. Future efforts may attempt to model the simultaneous decay of multiple markers ideally based on validation sets that include both mild and severe cholera infections. Second, our representative health seeking behavior survey results suggested that roughly one in four people with symptomatic cholera would seek care at one of the primary surveillance sites in Sitakunda for cholera like symptoms. This was based on the assumption that responses related to hypothetical moderate diarrhea represented the ‘average’ severity of disease, however, estimates of symptomatic incidence rates did vary  when care-seeking was based on alternative diarrhea severity definitions  47. Using the same PCR protocol, we additionally tested a random subset of nearly half of the RDT negative samples (46%). All diagnostic test results were used in a latent class model  located in the Chattogram district of Bangladesh. Two public health facilities provide care for diarrheal patients: the Sitakunda sub-district hospital  and the Bangladesh Institute for Tropical Infectious Diseases (BITID). These two sites are the primary sites for surveillance of diarrheal disease and cholera for the Bangladesh Directorate General for Health Services in Sitakunda. From January 24, 2021 through February 13, 2022, we surveilled the in-patient and out-patient wards of both health facilities, and attempted to enroll all suspected cases ≥1 years old presenting with non-bloody, acute watery diarrhea (3 or more loose stools in the 24-hours preceding the visit). After obtaining informed consent we administered a short, structured questionnaire and collected a stool  specimen for laboratory analyses. We tested each patient’s fecal sample onsite with the CholKit Rapid Diagnostic Test , then placed the sample in Cary Blair media and on Whatman 903 filter paper for subsequent lab testing at icddr,b in Dhaka. Due to the expected high sensitivity and moderate specificity of the RDT odel see to deter2 grid-cells and then randomly selected grid-cells proportional to the number of households in each with replacement. Within each selected grid-cell, we randomly selected structures (or GPS coordinates) weighted by whether they were classified as single- or multi-story units; the number of structures selected by grid-cell varied and only one structure was enrolled per GPS coordinate selected. If no structure was found at the point or the structure located at the point was not residential, the study team attempted to enroll the nearest residential household within 20 meters or proceeded to the next assigned point if no residential household existed within 20 meters. If the structure found was multi-story, the study team enumerated the number of residential households inside and generated a random number to determine which household to attempt to enroll. If no one was at the household once it was found, the study team attempted to revisit up to three times in the following 24 hours. If no one was at home after the attempted revisits or the household refused to participate, the study team proceeded to the next point (or attempted to enroll the next closest residence to the right if in a multi-story unit).We enrolled a population representative cohort in Sitakunda between 27-March-2021 and 13-June-2021, including a ~1-month gap due to a national COVID-19 related lockdown (referred to as rounds R0A & R0B throughout). To enroll households, we used two-stage sampling based on satellite imagery  with digitized building footprints classified as single or multi-story units, where we first divided the Sitakunda subdistrict into 1kmAfter receiving verbal consent from the head of household (or representative), we attempted to enroll all persons ≥1 year of age that were members of the household  and asked for written consent (and assent for those 7–17 years old) from each person. When household members that met this inclusion criteria were not present, we attempted to revisit up to three times in the survey period to enroll them. Survey staff administered a structured household-level questionnaire to the head (or representative) of each enrolled household. They also administered an individual-level questionnaire to and collected venous blood from each consenting participant (~5ml from adults and ~3ml from children <5 years old). The baseline individual-level questionnaire included questions on each persons’ demographics, mobility patterns, health status and healthcare seeking behaviors. Each enrolled household had two follow-up visits at approximately 4-month intervals (R1 from 21-September-2021 to 9-October-2021 and R2 from 25-January-2022 to 13-February-20221). At each follow-up visit, members of households who were not previously enrolled (or declined to participate in the previous round) were eligible to participate, and all consenting participants were administered a follow-up individual-level questionnaire, venous blood draw, and household heads a follow-up household-level questionnaire. Follow-up questionnaires were aimed at recent clinical disease episodes, healthcare seeking and WASH-related behaviors like individual drinking water sources and household sanitation and water infrastructure.To gauge people’s propensity to seek care for cholera, we asked a series of questions at baseline to each participant about three levels of hypothetical diarrhea severity: mild, moderate, and severe. We asked whether individuals would seek healthcare for each syndrome, and if yes, the specific type of care or facility  they would first visit. While the clinical spectrum of cholera is variable, we focused our main analyses on the self-reported care seeking for moderate-to-severe diarrhea (experiencing 3 or more loose stools in a day and dehydration) while exploring the other severities in sensitivity analyses.21, we tested each sample with the vibriocidal assay, with all samples from the same participant run on the same plate. Serum samples collected at different rounds were tested for vibriocidal antibody response against the homologous serotype of bacteria using the V. cholerae O1 El Tor Ogawa (strain 25049) or Inaba (strain 19479) strains as the target organism 48. Guinea pig complement  was used at a 1:10 dilution. The ODs of the plates were measured at 595 nm. A known pooled sera  was used in each plate as a control. The vibriocidal titer was defined as the reciprocal of the highest serum dilutions causing a greater than 50% reduction of the OD at 595 nm when compared with the OD of the control wells without serum. Wells containing physiological saline, and growth medium were included on each plate to exclude the possibility of bacterial contamination of the reagents.Blood samples were centrifuged the day of collection and frozen at −80C until the time of analysis (1–11 months). Following previously described methods 49. We extrapolated population counts by age group and sex assuming 1.5% annual population growth for the 10 years between 2011 and 2021. Since children under one year of age were excluded in the survey and clinical surveillance, we subtracted 20% of the total population for the 0–4 age group, following the age distribution reported in the US Census Bureau International Database for Bangladesh in 2021 50.We used the reported population size by 5-year age bins for Sitakunda from the 2011 National Census46, and in some cases PCR and/or culture, which are also imperfect 51, we modeled the array of test outcomes using a multinomial likelihood, marginalizing over potential outcomes of tests not performed while accounting for the conditional dependence between the tests inherent in study design  with priors on performance derived from a prior study in Bangladesh 51. The result of this model component is the posterior distribution of the age-group specific daily incidence rate of cholera and non-cholera AWD from study health facilities. Complete details of this model are provided in the We estimated the age-stratified incidence rates of medically attended cholera and non-cholera suspected cases within our study facilities using a Bayesian model fit to data from systematic laboratory testing of all suspected cases. Data were restricted to individuals residing in Sitakunda subdistrict visiting the healthcare facilities from the start of the study period until February 13, 2022, the end of the last serosurvey. In short, we assume that the incidence rate of acute water diarrhea  any rise in vibriocidal titer between visits or (b) any rise greater than a threshold , these will be biased as titers decay quickly, often within months, and thus in a shorter time frame than our study visits. Also, these potential definitions do not account for measurement error in the vibriocidal assay. We, therefore, developed a Bayesian inference framework to estimate the seroincidence rate in the population based on serial measurements accounting for the decay kinetics of vibriocidal antibodies, the observed measurement error in the assay and the reconstructed epidemic curve of medically attended cholera cases as a proxy for the force of infection experienced by study participants. We restricted these analyses to only those who participated in all three study visits .Our estimates of infection incidence are based on serial measurements of vibriocidal antibodies developed in response to 22. In this model, vibriocidal antibodies were assumed to have an instantaneous boost after some delay post-infection from their baseline levels and then decay exponentially at a constant rate. The differences (or lack thereof) in vibriocidal measurement between rounds could indicate (1) absence of recent infection with any differences due to measurement error, (2) prior infection (before the last study visit) leading to a decay in vibriocidal titers, or (3) an infection at some unknown time since the last visit potentially with some decay in titer levels. Our inference framework estimates the likelihood of each of these possibilities for each person-sample, while utilizing the data from clinical surveillance to infer the likelihood of infections at each timepoint between measurements. The likelihood function is based on eight potential outcomes for individual-level infection status before baseline and between each study round for each participant . We can then estimate the probability of infection between any two arbitrary time points  Supplement 1


Preventive interventions that are effective in reducing the incidence of mental disorders in adolescence and early adulthood may impact substantially on lifetime economic, educational, and health outcomes; however, relatively few studies have examined the capacity of alternative approaches to preventing youth mental disorders  to reduce disorder incidence at a population level. Using a dynamic model of the onset of non-specific, relatively mild symptoms and progression to more severe disease, we show that: (1) indicated preventive interventions, targeting adolescents and young adults experiencing subthreshold symptoms, may often be more effective in reducing mental disorder prevalence than universal interventions delivered to the general population ; and (2) the ability of selective preventive interventions  to alter the prevalence of mental disorders is severely restricted by an inverse relationship between the prevalence of significant risk factors for mental illness and the relative risk of developing symptoms. Nearly two-thirds of people with a mental disorder (62.5%) first develop symptoms as adolescents or young adults (before the age of 25), with a peak age of onset of 14.5 years (median age of onset 18 years)2. Adolescence and early adulthood are critical periods for establishing the social, cultural, emotional, and educational resources underpinning both the successful adoption of adult roles and responsibilities  and the maintenance of health and wellbeing into middle and late adulthood3. Prospective analyses indicate that the development of a serious mental disorder during this pivotal stage of life is a significant predictor of persistent, adverse socioeconomic and health outcomes, including economic non-participation, unemployment, low income, welfare dependency, low educational attainment , and poor physical health6. Nevertheless, available evidence also indicates that relatively mild mental disorders originating in adolescence frequently do not persist into early adulthood7, so that effective, youth-focussed interventions designed to reduce the risk of symptom onset or prevent progression from relatively mild to more severe mental health problems have the potential to impact substantially on longer-term economic, educational, and health outcomes.Mental disorders are a principal cause of disability globally, directly affecting an estimated 12.6% of the world population and accounting for 14.6% of years lived with disability 9, namely (in order of increasing risk): (1) universal preventive interventions, which are considered appropriate for a general target population defined on the basis of some criterion unrelated to the risk of mental illness ; (2) selective preventive interventions, targeting asymptomatic individuals exposed to one or more biological, psychological, or socioeconomic risk factors that significantly increase their risk of developing a mental disorder ; and (3) indicated preventive interventions, which aim to prevent progression to full-threshold disorders 11 in individuals presenting with non-specific, relatively mild symptoms (early stage 1), typically through the provision of psychological and social support. Although many studies have examined the individual-level effectiveness of particular universal, selective, and indicated interventions in preventing mental disorders in adolescents and young adults12, the potential impacts of these alternative approaches to prevention on population mental health  have received considerably less attention. This paper presents a dynamic modelling analysis of the general conditions under which each type of intervention  would be expected to significantly reduce the incidence of mental disorders in adolescence and early adulthood, providing a framework for assessing the potential population-level impacts of specific preventive interventions and, more broadly, for guiding the development of effective prevention strategies.Preventive interventions for mental disorders can be divided into three principal types according to the average risk of developing a disorder among individuals in the target populationThe dynamic model developed for the analyses Fig.  compriseNon-vulnerable and vulnerable youth develop symptoms at rates (per year) equal to 15. Accordingly, the equilibrium prevalence values calculated for different combinations of values Panels A–C of Fig. entclass1pt{minimaFigure c. 0.1 or less), so that the population-level effects of selective preventive interventions will typically be restricted to those indicated in the upper panels and lower left panel of Fig. 19. Assuming values of 0.4 for While the results presented in Fig. entclass1pt{minima20, indicated prevention will typically produce greater population-level effects than universal and selective approaches (at least for available interventions). And thirdly, substantial increases in the population-level effectiveness of multi-component prevention programs may be achieved via increases in the individual-level effectiveness of universal and indicated preventive interventions or, to a lesser extent, by improving risk prediction models  disorders will occur among individuals at relatively low risk of developing symptoms simply because these individuals comprise most of the population, so that approaches to prevention focussing on the smaller number of individuals at very high risk (the ‘high-risk’ strategy) may often be less effective in reducing disorder prevalence than approaches aimed at decreasing the mean level of risk for the population as a whole (the population strategy). The results presented here for universal and selective preventive interventions clearly demonstrate the potential for whole-population approaches  to deliver greater population-level effects than targeted approaches; where the proportion of adolescents and young adults at significantly increased risk of developing symptoms is low .According to Rose’s 22. The effectiveness of a multifaceted prevention program in reducing the burden of youth mental disorders will depend principally on the individual-level effectiveness of its component universal and indicated interventions and, to a more limited extent, on the reliability with which the risk of symptom onset can be predicted for vulnerable adolescents and young adults 24.Despite the focus of our analysis on evaluating and comparing the population-level impacts of single preventive interventions, we assume that any comprehensive prevention strategy capable of substantially reducing not only the prevalence of more severe mental disorders, but also the incidence of relatively mild, subthreshold symptoms, demand for (and therefore pressure on) mental health services, and social and economic inequalities in disorder prevalence, will necessarily include a mix of evidenced-based universal, selective, and indicated interventionsase Fig. , so that8). Analyses of more complex dynamic models (particularly agent-based models)26 are needed to determine the degree to which the individual-level variability of these factors may affect our conclusions. Secondly, the values assumed for the parameters 27 yield results qualitatively similar to those presented in Fig. There are several limitations of the analysis presented here that should be pointed out. Firstly, our model only captures aggregate  dynamics, and so effectively disregards potentially significant heterogeneity in individual-level factors that are expected to influence the incidence of subthreshold symptoms, engagement with services, progression to more severe disorders, and responses to specific preventive interventions  than universal preventive interventions20, so that indicated prevention may often produce greater population-level effects than universal prevention, at least for currently available interventions. An inverse relationship between the prevalence of significant risk factors for mental illness and the relative risk of developing subthreshold symptoms (values of In conclusion, the dynamic modelling analysis presented here indicates that the ability of a multifaceted prevention program to reduce the incidence of youth mental disorders will depend primarily on the individual-level efficacy of its component universal and indicated interventions see Fig. . SignifiPopulation-level effects for alternative preventive interventions were assessed by determining the equilibrium prevalence of full-threshold mental disorders under scenarios in which a focal intervention with constant individual-level efficacy is introduced into the population and remains in place indefinitely (see panel A of Fig. mentclass2pt{minimmentclass2pt{minimSupplementary Information.


Monogenic intestinal epithelial disorders, also known as congenital diarrheas and enteropathies (CoDEs), are a group of rare diseases that result from mutations in genes that primarily affect intestinal epithelial cell function. Patients with CoDE disorders generally present with infantile-onset diarrhea and poor growth, and often require intensive fluid and nutritional management. CoDE disorders can be classified into several categories that relate to broad areas of epithelial function, structure, and development. The advent of accessible and low-cost genetic sequencing has accelerated discovery in the field with over 45 different genes now associated with CoDE disorders. Despite this increasing knowledge in the causal genetics of disease, the underlying cellular pathophysiology remains incompletely understood for many disorders. Consequently, clinical management options for CoDE disorders are currently limited and there is an urgent need for new and disorder-specific therapies. In this review, we provide a general overview of CoDE disorders, including a historical perspective of the field and relationship to other monogenic disorders of the intestine. We describe the genetics, clinical presentation, and known pathophysiology for specific disorders. Lastly, we describe the major challenges relating to CoDE disorders, briefly outline key areas that need further study, and provide a perspective on the future genetic and therapeutic landscape.The online version contains supplementary material available at 10.1007/s00439-022-02501-5. Diarrhea and malabsorption in infancy are a relatively common occurrence and can usually be ascribed to a variety of acquired or maternally derived causes such as congenital infections and food protein intolerance. Monogenic intestinal epithelial disorders, also known as congenital diarrheas and enteropathies (CoDEs), are a rare cause of severe, life-threatening diarrhea, and describe a heterogenous group of disorders that result from single gene variants that directly alter epithelial function. These disorders are characterized by neonatal or infantile-onset diarrhea and malabsorption and in general require intensive medical support including intravenous nutrition and fluids. Many of these disorders are associated with altered function in other organ systems, including the immune system; however, disruption of intestinal epithelial structure and function are a universal feature that define this group of genetic disorders. Here, we provide an overview of CoDE disorders, including the advances in their diagnosis and cell biology, placing them in context with other genetic disorders causing intestinal disease. We review in detail archetypal CoDEs as well as recently discovered genes and outline some of the major challenges and opportunities in the field.A brief history of CoDE disorders: Infants who exhibit severe and persistent intestinal fluid loss and malabsorption requiring intensive care level support from birth or in the first few weeks of life are a rare event. These infants often require a high level of support to maintain life and to undergo further evaluation and diagnosis. Prior to the advent of the genomic era in research and clinical medicine, infants who survived past the neonatal period were often grouped together with a diagnosis of “intractable diarrhea of infancy”. This term was popularized by Avery et al. in their description of series of infants with chronic diarrhea of unknown etiology  for glucose-galactose malabsorption  for congenital chloride diarrhea  or FOXP3 mutations (IPEX) that primarily affect immune cell function, or development  and finger-like projections (villi). Intestinal epithelial cells (IECs) are generated from pluripotent stem cells that reside in the base of the crypts and are marked by the receptor protein Lgr5. These stem cells give rise to progenitor cell populations that ultimately differentiate into a wide range of different epithelial subtypes including secretory cells such as goblet cells, and enteroendocrine cells, as well as columnar absorptive cells  and digestive enzymes  . This is particularly relevant for current and future studies that aim to correlate specific mutations with phenotype, prognosis, and treatment. As with any attempt to classify such a heterogenous group of disorders there are specific genes/disorders that do not fit well in any one category. In some cases, such as the recently discovered WNT2B deficiency, this has allowed expansion or development of new categories. As new causative genes  and new information on disease mechanisms continue to emerge, these categories will need ongoing revision and refinement. Disease can also be classified by other methods such as protein ontology  or clinical outcome .Although they are commonly linked and defined by altered intestinal epithelial function, CoDE disorders exhibit a wide variety of cell and tissue level pathophysiological mechanisms. Despite this heterogeneity some broad categories have emerged to classify specific genes. As the specific causative genes for many cases of non-acquired severe infantile-onset chronic diarrhea can now be established, it has become important to shift away from syndromic or clinically defined naming of disorders to more specific designation by affected gene  either directly via loss-of-function mutations in NHE3 or indirectly via gain-of-function mutations in the receptor GUCY2C. GUCY2C mutations also cause activation of chloride secretion via apical membrane chloride channels. Nutrient dependent sodium absorption is altered in glucose-galactose malabsorption via SGLT/SLC5A1 and loss of sodium-dependent bile transport in the ileum leads to congenital bile acid mediated diarrheas.Perhaps the most archetypal of CoDE disorders are those that involve alterations in intestinal epithelial transport function. Several disorders affect the process electroneutral sodium absorption and parallel chloride/bicarbonate handling (SLC26A3) gene is mapped to chromosome 7q31 and encodes for the chloride/bicarbonate exchanger, also known as down regulated in adenoma (DRA), a transmembrane protein localized mainly to the apical side of the mucosa in the ileum and proximal colon  exchanger, encoded by SLC26A3, plays a major role in fluid homeostasis in the human body. It has the task of transporting chloride ions in exchange for bicarbonate across the apical membrane of intestinal epithelial cells. This drives electroneutral sodium absorption through coupled Na+/H+ exchangers (NHE3) in the intestine  later in life. This is supported by mouse studies of Slc26a3 deficiency that show increased susceptibility to induction of colitis , localized primarily on the apical side of intestinal and proximal renal tubular epithelial cells. NHE3 is responsible for the bulk of electroneutral Na+ luminal absorption in the small and large intestine in exchange for intracellular protons, as well as for most reabsorption of filtered Na+ in the proximal renal tubules  associated with ulcerative colitis, suggesting a potential explanation for the association between SLC9A3 mutations and the development of inflammatory bowel disease later in life , localized to the apical membrane of enterocytes and activated by guanylin, uroguanylin, and the heat-stable Escherichia coli enterotoxin heat-stable enterotoxin (STa). Activating mutations, which are inherited in an autosomal dominant manner, are a recognized cause of non-syndromic secretory congenital sodium diarrhea , reducing sodium absorption and together with elevated chloride secretion result in fluid loss and diarrhea , a 73 k-Da transmembrane protein, which is present mainly in the small intestine, the heart, and the kidneys  and the sodium-dependent glucose transporters (SGLT)  and key proteins in intestinal fat processing such as diacylglycerol O-acyltransferase 1 (DGAT1). A very novel member of this group is SI) gene, mapped to chromosome 3q26.1, codes for sucrase-isomaltase, a heterodimeric protein with two subunits, sucrase and isomaltase. Sucrase-isomaltase is a type II transmembrane disaccharidase glycoprotein expressed in the intestinal brush border , was initially identified and described in the 1960s  is a microsomal enzyme that plays a critical role in synthesizing cellular triacylglycerols Fig. , using dDGAT1 deficiency reveals mild villous blunting and chronic inflammation with eosinophilia in the lamina propria  and one in-frame deletion (p.Ala226_Arg250del) have been identified  and homozygous hypobetalipoproteinemia (HHBL) are syndromes resulting from the total absence or extremely low levels of apolipoprotein B. These patients present similarly during infancy with fatty diarrhea and associated fat-soluble vitamin malabsorption , a protein which heterodimerizes with protein disulfide isomerase (PDI) and loads lipids onto apolipoprotein B  and 2152 amino acids in its splice variant form (apoB-48)  with supplementation of essential fatty acids and fat-soluble vitamins Welty . EvaluatSAR1B gene, which encodes for the Sar1b protein, are mapped to chromosome 5q31.1 and are implicated in the development of chylomicron retention disease (CRD), now presumed to be the same disorder as Anderson disease. SAR1B deficiency is characterized by severe fat malabsorption, failure to thrive, diarrhea, and vomiting in infancy or childhood. Consequently, patients affected by a variety of loss-of-function variants of the SAR1B gene present with low blood cholesterol levels, absent chylomicrons, deficiency of cholesterol-dependent fat-soluble vitamins, and subsequent neurological impairment.Mutations of the The presentation of CRD on esophagogastroduodenoscopy (EGD) is quite striking, with authors describing white duodenal and jejunal mucosa , a key player in the pancreatic enzyme activation cascade. The link between malabsorption and enterokinase deficiency (EKD) was first established by Hadorn et al. in 1969, who identified an infant with chronic malabsorption and low proteolytic enzyme activity which could be corrected by the addition of exogenous enterokinase in vitro  which helps regulate protein folding and disulfide bridging in the endoplasmic reticulum and may also be secreted to interact with the extracellular matrix independent of its PDI activity , key to our ability to digest lactose in milk, is a brush-border β-galactosidase encoded by the Small intestinal biopsy of patients with CLD demonstrates normal histology without villus atrophy or inflammation, with low or absent lactase activity  and Tufting Enteropathy (EPCAM). Several genes involve polarized trafficking, which in epithelial cells relies on unique vesicular compartments not always present in other cells. The epithelial apical recycling pathway involving Myo5B powered vesicular trafficking has been found to be affected by mutations to multiple different genes , pointing to the importance of this pathway in normal epithelial function. Congenital disorders of glycosylation involving the intestinal epithelia are a well-known but poorly understood cause of intestinal dysfunction. Lastly, mutations in genes such as TTC7A and SPINT2 cause interesting structural and developmental defects in epithelial cells. Gene loss-of-function studies in many of these disorders have revealed fascinating and deep insights in the fundamental cell biology of intestinal epithelial cells.Genes involved in epithelial structure, trafficking and polarity comprise an important group of CoDE disorders, and include classical disorders such as Microvillus Inclusion Disease , and the literature demonstrates a strong correlation between mutation characteristics and disease phenotype  was first described in a 1978 case series of five infants by Davidson et al. The infants presented with persistent diarrhea from birth and histopathologic findings of villous atrophy, crypt hyperplasia, and “lysosome-like inclusions” , and organized microvilli are the histopathologic hallmark of the disease , ATP binding (p.Gly168Arg), ATP hydrolysis (p.Arg219His), motor domain mechanics , and protein folding   were found in patients with familial hemophagocytic lymphohistiocytosis type-5 (HLH5) by zur Stadt et al. in 2009  is necessary for proper cytotoxic cell degranulation, while it appears that STXBP2 interaction with STX3 is necessary for proper apical recycling , eight of whom were found to have severe retinal dystrophy  is a t-SNARE protein which is vital for integration of the recycling endosome into the apical plasma membrane of intestinal epithelial cells  syndrome  is a myosin co-chaperone required for adequate protein folding and expression of myosin Vb, among other myosins  domain, a central domain, and a C-terminal UCS domain. The TRP domain appears to be involved in the recruitment of heat shock protein-family chaperones Hsp70 and Hsp90 while the UCS domain has been shown to be critical for myosin binding   is a zinc-dependent metalloenzyme which catalyzes the transformation of fructose-6-phosphate (F6P) to mannose-6-phosphate (M6P)  type 1b is characterized by partial villous atrophy with mild lymphangiectasia, while liver biopsy in these patients demonstrates congenital hepatic fibrosis. Ultrastructural analysis of hepatocytes in CDG type 1b is notable for lysosomal inclusions, presumably due to the accumulation of mis-trafficked proteins  or localize to the beta sheets which make up the core of the protein , with some localizing to the C-terminal domain   is a subunit of the adaptor protein-1 complex (AP-1), which is necessary for clathrin coat assembly at the trans-Golgi network and endosomes Duncan . This suKnockout of AP1S1 or sole expression of patient-derived variants p.Leu90Pro and p.Glu116Lys in CaCo-2 cells results in preferential mis-localization of ZO-1 and claudin-3 to the basolateral membrane , also referred to as “syndromic diarrhea” or “phenotypic diarrhea of infancy”, is a multisystemic disease that is inherited in an autosomal recessive fashion and affects around 1 in 1,000,000 live births  and p.Trp1524* (which is found in families from Turkey)  protein, which is normally expressed on the basolateral membrane of cells in epithelial tissues and on plasma cells , also known as intestinal epithelial dysplasia, presents in the first days of life with life-threatening intractable watery diarrhea, electrolyte disturbances, and failure to thrive  is a negative regulator of multiple endogenous serine proteases. Disease-associated SLC9A3 and described in the section on disorders of epithelial transporters. In the healthy intestinal epithelium, serine proteases regulate both ENaC and EpCAM. The membrane-anchored serine proteases prostasin and matriptase are converted to their active forms by reciprocal zymogen activation, linking their functionality. Prostasin and matriptase both potently activate ENaC by cleaving in the γ-subunit and increasing the channel’s open probability  was discovered to be the causative gene for congenital disorder with a spectrum of abnormalities including multiple intestinal atresias (MIA), enteropathy, intestinal inflammation, variety of skin and hair manifestations, and a variable severity combined immunodeficiency. In parallel in 2013, three groups reported TTC7A mutations in cohorts of patients with intestinal atresias, combined immunodeficiency and intestinal inflammation. Samuels et al. demonstrated the presence of TTC7A lead to profound abnormalities in intestinal epithelial polarity and lumen formation. This results in a highly architecturally distorted epithelium particularly in the colon characterized by stratified or pseudostratified epithelium, formation of crypt glands with multiple lumens, and mis-localization of apical and basolateral markers  repeats. It has been shown to be important in the membrane localization and generation of phosphoinositides by chaperoning phosphatidylinositol-4-kinase (PI4K)-IIIα to cellular membranes  stem from genes largely involved in normal enteroendocrine-cell development / differentiation resulting in a loss of this cell population and provided insights on their importance for normal fluid and nutrient absorption.Enteroendocrine cells are a critical secretory epithelial cell population in the intestine that play a variety of regulatory and sensory roles. Several CODE disorders (NEUROG3) is a gene located in chromosome 10q22.1 which plays a critical role in the differentiation of enteric stem cells and pancreatic progenitor cells. Homozygous missense mutations on this gene were first described to be causative of human disease in 2006 in a group of patients who presented with intractable malabsorptive diarrhea after initiation of feeds , a basic helix–loop–helix transcription factor with a well-established role in neurogenic development. Studies involving mice have proven that neurogenin-3 is essential for the differentiation of all pancreatic endocrine precursors . PERCC1 was very recently found to be the causative gene in a series of cases with unknown infantile-onset malabsorptive diarrhea and failure to thrive. Uniquely, the discovery of loss of PERCC1 function was made by analysis of a non-coding region of the genome found commonly deleted in the case series ”, a non-coding sequence that was subsequently found to control expression of PERCC1. This finding came after eight patients of Jewish Iraqi origin who presented with severe congenital malabsorptive diarrhea were found, by whole-exome and whole-genome sequencing, to have a homozygous deletion of the ICR. The ICR was defined as the minimal 1,528 base pair overlapping region between the homozygous 7,013-bp deletion and the compound heterozygous 3,101-bp deletion. As expected, analysis of single-cell transcriptomes from the murine intestine identified Percc1-positive cells as enteroendocrine, with expression of Sox and Ngn3. Analysis of RNA sequencing data from Percc1 mouse knockout models demonstrated significant stomach and intestinal downregulation of many hormones dependent on enteroendocrine-cell production, such as gastrin and somatostatin, with upregulation of certain proinflammatory genes. Interestingly, human intestinal organoids generated from affected patients initially showed no morphological abnormalities, but later, by day 42, a significant decrease was seen in the number of enteroendocrine cells in PERCC1 patient-derived organoids. This suggests that the role of PERCC1 may occur later in enteroendocrine development.Recently, computational analysis of the PERCC1 protein sequence has shown that PERCC1 is a YAP (Yes-associated protein), TAZ , and FAM181 homologue; these transcriptional regulators bind to DNA through TEADs (TEA/ATTs domain) . A calcium-dependent serine endoprotease required for the biosynthetic processing of hormone and neuropeptide precursors and their transformation into their fully functional forms  point mutation in exon 8 of the PCSK1 gene  was reported with the described missense variant predicted by homology modeling to disrupt post-translational acylation of the protein  remains to be explored and are the subject of ongoing studies.The addition of this case also enabled confirmation of the gastrointestinal phenotype and the extension of phenotype to other organs, most notably the eye. Three out of the four reported patients were also found to have significant anterior chamber abnormalities  establishing Dyskeratosis congenita (DC) is a multisystem disorder caused by impaired telomere maintenance. The hallmark of DC is the clinical triad of dysplastic nails, lacy reticular pigmentation of the upper back or neck, and oral leukoplakia (Savage and Niewisch TERT gene) forms a multi-protein complex at the ends of chromosomal DNA  in 1998 . The EGF signaling pathway has been well characterized as a vital mitogenic signaling pathway which supports and regulates the stem cell niche in many organs, including the gut  is becoming increasingly utilized by clinicians in many specialties, as testing becomes progressively more available and turnaround times for results become shorter. As the biotechnology industry races to advance technologies necessary for widely available genetic sequencing, the cost of sequencing a genome has dropped from 100 million US dollars in 2001 to less than 1000 US dollars in 2021, outstripping the pace of even Moore’s Law for computer processing power  has now brought together many of these community resources and an important next step for the field is to leverage these tools for therapeutic benefit.As the genetic basis of CoDE disorders becomes increasingly more defined and precise, the next major challenge in the field relates to the development of therapies for these rare disorders. A critical first step in this process is understanding the tissue pathophysiology, genotype–phenotype correlations and cell biology related to the epithelial proteins involved. Studies on the natural history of individual disorders and in-depth analysis of tissue histology are needed including the use of new spatial methods such as multiplex immunofluorescence  represent a particularly attractive option for this application, as they can be designed upon the basis of the coding sequence alone. ASOs are modified nucleotide sequences which are capable of changing protein expression by interacting with immature mRNA to either promote mRNA degradation or to affect mRNA splicing Below is the link to the electronic supplementary material.