Neuroglial cells have a high level of plasticity, and many types of these cells are present in the nervous system. Neuroglial cells provide diverse therapeutic targets for neurological diseases and injury repair. Cell reprogramming technology provides an efficient pathway for cell transformation during neural regeneration, while transcription factor-mediated reprogramming can facilitate the understanding of how neuroglial cells mature into functional neurons and promote neurological function recovery. The ter2In the nervous system, all methods of transforming non-neuronal cells into neurons are presently caused damage to brain, and the emergence of cell reprogramming technology may allow non-neuronal cells to produce a variety of specific cell types, including neurons . In cellNeuroglial cells play a variety of roles in the physiological and pathological processes of the central nervous system, such as maintaining homeostasis, providing neurotrophic proteins to neurons and regulating nerve signaling. Recently, there has been increasing evidence that glial cells can also act as nerve stem/ progenitor cells and contribute to adult neurogenesis or nerve regeneration. For example, astrocytes and oligodendrocyte precursor cells may be activated to proliferate and differentiate under pathological conditions. When cultured in vitro, they can form neural spheres capable of differentiating into astrocytes, oligodendrocytes and neurons. In addition, forced expression of exogenous genes in astrocytes and NG2-glia successfully reprogrammed them into neurons, which may also indicate their stem/progenitor characteristics . Niu’s tThus, direct reprogramming is caused by different transcription factors in specific cell lines and epigenetic backgrounds .3Astrocytes exhibit two states: static and reactive. These states differ in that static astrocytes cannot divide but can proliferate and differentiate when activated by injury states  or otherNeurogenic factors and REST-assisted inhibitors  regulate the transcriptional mechanism of astrocyte reprogramming into neurons. Masserdotti et al  found thZhao et al  suggesteIn addition to demonstrating that reactive astrocytes or astrocytes in the brain of postnatal mice can be directly reprogrammed into neurons or stem cell-like cells by overexpression of certain transcription factors, induced neuronal stem cells (iNSCs) can also be derived from astrocytes in vivo. Huang and colleagues  demonstrOligodendrocyte precursor cells (OPCs) can develop into multipotent neuron-like stem cells (NSLCs), which can self-renew and produce neurons, astrocytes and oligodendrocytes when exposed to certain extracellular signals. This was an important finding in developmental neurobiology, suggesting that glia have the potential to be reprogrammed into stem cells for treatment of neurological diseases . Externa4Myelination of the PNS is essential for maintaining axonal function. After peripheral nerve injury, the function of SCs changes from myelination to demyelination. SC reprogramming affects the survival and demyelination of neurons and their supporting axons. This reprogramming is regulated by distal regulatory elements, which also integrate multiple transcription factors .SCs can repair injured neurons, facilitate their survival and provide necessary pathways for axon regeneration and target innervation. The transformation of reparative SCs involves dedifferentiation and alternative differentiation or activation, commonly referred to as (direct or pedigree) reprogramming. Damage-induced SC reprogramming involves activation of myelin genes and a series of repair factors, including up- regulation of nutrient factors, elevation of cytokines involved in the immune response, activation of the myelin sheath to halt myelin autophagy and macrophage aggregation in SCs and determination of the regeneration trajectory of the target through axon orientation. The repair process involves the regulatory mechanism of the transcription factor c-Jun. After injury, c-Jun is rapidly up-regulated in SCs. In contrast, in the absence of c-Jun, injury leads to dysfunction, neuronal death and functional recovery failure. Therefore, although c-Jun is not necessary for SC development, it is essential for SC reprogramming and post-injury repair . c-Jun lIn SC dedifferentiation, negative regulators of myelin formation can offset the positive regulators of myelin formation in most cases and may play a major role after nerve injury. For example, the transcription regulators SOX2, Pax3, ID2, EGR1 and EGR3 can mediate SC dedifferentiation in damaged nerves. In vitro, overexpression of SOX2, Pax3 and ID2 can reduce the expression of the myelin gene . In addiNOTCH is a transmembrane receptor protein that is lysed after ligand binding to produce an intracellular domain. As a transcriptional regulator, NOTCH controls the proliferation of SCs and promotes the production of immature SCs by SCPs in vivo, but also acts as a negative regulator of myelin formation. During the formation of the myelin sheath, the NOTCH level gradually decreases, and its inactivation or over- activation will lead to premature or delayed myelin sheath formation, respectively . ZEB2 coFurthermore, the binding sites of the transcription factors SOX10 and EGR2/KROX20 cannot be identified by the activity enhancer recognition marker h3k27ac, which is the key determinant of SC differentiation. The expression of the transcription factor EGR2 disappears after nerve injury. h3k27a can accurately indicate the dynamic changes in SC transcription, and it can be used to monitor the effect of transcription factors on myelin formation in SCs and the response of the PNS to nerve injury .Satellite glial cells are a group of cells around primary sensory neurons that are similar to astrocytes in the central nervous system . As a ty5A study published in the Journal Nature Biotechnology showed that reprogramming astrocytes into dopaminergic neurons improves motor function in mice with Parkinson’s disease (PD). This method can be used in place of traditional cell replacement. In mice with unilateral striatal astrocyte deletion (a PD model), lentiviral injection induced the overexpression of NeAL218 . At 5 weeks after lentiviral induction of NeAL218, dopaminergic neurons were increased, and motor function had recovered. This in situ reprogramming of astrocytes for PD provides a new possibility for cell replacement therapy, thus avoiding unnecessary cell transplantation or immunosuppression . RivettiReactive glial cells produced after brain injury in mice or in AD models can reprogram functional neurons directly in vivo through the transcription factor NEUROD1. NEUROD1 mediates reprogramming of astrocytes into glutamatergic neurons and the reprogramming of NG2 glial cells into glutamatergic neurons and GABAergic neurons. This process of reprogramming glial cells into different types of neurons using the same transcription factor may provide important clues to the lineage relationship between neurons and glial cells. Reactive glial cells can be reprogrammed as functional neurons in the brain of injured or diseased mice, which may provide a treatment for reactive gliosis, which is widely associated with nerve injury and neurodegenerative diseases. In situ reprogramming of functional neurons by reactive astrocytes and NG2 cells may provide new research topics for brain repair via internal reprogramming of neurons . However6In conclusion, transcription factor-mediated neuroglial reprogramming is a promising field in the study of cell replacement therapy in the nervous system. At present, researchers can directly reprogram astrocytes to form neuronal cells in vivo and in vitro. Compared with in vitro methods, in vivo reprogramming eliminates the process of cell culture and transplantation, which has the benefit of allowing cells to be regulated by transcription factors and other factors that affect cell survival, leading to precise reconstruction of endogenous cells, tissues and organs . It is aCompared with the CNS, the study of neuroglial cell reprogramming in the PNS is at an early stage. The regeneration ability of the PNS is mainly related to the plasticity of SCs. Therefore, transcription factors involved in myelin formation provide a new therapeutic strategy for cell reprogramming in the case of peripheral nerve diseases or peripheral nerve injuries. Many researchers continue to optimize reprogramming techniques to use stem / progenitor-like glial cells as endogenous cells to repair nerves . Current


What are the common presenting symptoms of cancer in adolescents and young adults aged 12 to 24 years and how are they associated with help-seeking?This cross-sectional analysis of 803 adolescents and young adult patients found a range of presenting symptoms in diverse combinations, which varied by cancer group; 27% of patients waited more than 1 month to seek help for their symptoms.These findings suggest that adolescents and young adults with cancer present with a broad spectrum of symptoms, often at substantially higher proportions than previously reported; thus, re-examination of symptom prevalence in this patient group is warranted. This cross-sectional study examines the common presenting symptoms of cancer in adolescents and young adults aged 12 to 24 years and the potential variation in time to help-seeking by presenting symptom. Evidence regarding the presenting symptoms of cancer in adolescents and young adults can support the development of early diagnosis interventions.To examine common presenting symptoms in adolescents and young adults aged 12 to 24 years who subsequently received a diagnosis of cancer and potential variation in time to help-seeking by presenting symptom.This multicenter study is a cross-sectional analysis of the BRIGHTLIGHT cohort study, which was conducted across hospitals in England. Participants included adolescents and young adults aged 12 to 24 years with cancer. Information on 17 prespecified presenting symptoms and the interval between symptom onset and help-seeking  was collected through structured face-to-face interviews and was linked to national cancer registry data. Data analysis was performed from January 2018 to August 2019.Self-reported presenting symptoms.The main outcomes were frequencies of presenting symptoms and associated symptom signatures by cancer group and the proportion of patients with each presenting symptom whose patient interval was longer than 1 month.The study population consisted of 803 adolescents and young adults with valid symptom information . The number of symptoms varied by cancer group: for example, 88 patients with leukemia (86%) presented with 2 or more symptoms, whereas only 9 patients with melanoma (31%) presented with multiple symptoms. In total, 352 unique symptom combinations were reported, with the 10 most frequent combinations accounting for 304 patients (38%). Lump or swelling was reported by more than one-half the patients . Other common presenting symptoms across all cancers were extreme tiredness , unexplained pain , night sweats , lymphadenopathy , and weight loss . The relative frequencies of presenting symptoms also varied by cancer group; some symptoms (such as lump or swelling) were highly prevalent across several cancer groups . More than 1 in 4 patients (27%) reported a patient interval longer than 1 month; this varied from 6% (1 patient) for fits and seizures to 43% (18 patients) for recurrent infections.Adolescents and young adults with cancer present with a broad spectrum of symptoms, some of which are shared across cancer types. These findings point to discordant presenting symptom prevalence estimates when information is obtained from patient report vs health records and indicate the need for further symptom epidemiology research in this population. Patient-reported data could amplify our understanding of presenting symptoms as approaches based on the examination of patient EHRs rely on complete and accurate elicitation and recording of symptom history during clinical encounters.21 Therefore, we aimed to examine the nature and frequency of presenting symptoms and to describe their associated time to help-seeking in adolescents and young adults with a subsequent diagnosis of cancer, using novel self-reported data from a large patient cohort in England.Studying the presenting symptoms of adolescents and young adult cancers can help us understand time to help-seeking and associated variation among adolescents and young adults with symptoms who subsequently receive a diagnosis of cancer. However, there is limited evidence regarding the presenting symptoms of adolescents and young adults with cancer. Prior research22 This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The BRIGHTLIGHT study was approved by the London-Bloomsbury Research Ethics Committee and the National Health Service Health Research Authority Confidentiality Advisory Group. Participants gave written informed consent to take part in a face-to-face survey and for clinical information to be extracted from their EHRs; dates of diagnosis were obtained from national cancer registration . Participants younger than 16 years gave their assent in order to participate after consent was obtained from their parents or guardians. Those unable to complete the survey, unable to give consent, in custodial care, or facing imminent death were not eligible to take part in the study.22 A total of 1114 young people aged 12 to 24 years at diagnosis with any cancer were recruited between July 1, 2012, and April 30, 2015, from 96 nationwide English National Health Service Trust hospitals; 830 participants completed the baseline survey.We analyzed cross-sectional patient-level data from the BRIGHTLIGHT cohort.23; the survey consists of 15 domains identified by young people as important in their cancer experience, including their experience before diagnosis. The survey questions were mainly read out by the interviewer and answered from a prespecified list, with options for including free text and dates where applicable.Information on events and intervals between symptom onset and cancer diagnosis was collected through a structured face-to-face interview conducted with an independent survey provider (Ipsos MORI) using a device with computer-assisted personal interviewing software, which allows the interviewer to input data into a tablet. This has the benefit of having built-in methods for checking errors and allows routing of questions, automatically eliminating irrelevant questions on the basis of previous answers. The survey instrument development has been described previously23 Symptoms derived from the free-text information were either recoded as 1 of the 16 specified symptoms, or otherwise collated into a 17th symptom category labeled as other symptoms (eTable 1 in the Information on symptoms experienced before the diagnosis of cancer was ascertained through yes-or-no responses to 16 specified symptoms (patients could respond yes to >1 symptoms), with any other symptoms recorded as free-text responses.12 Participants were asked, “From the time when you first noticed a symptom of cancer how long was it before you saw the [general practitioner or accident and emergency]?” and they were provided with 1 of the following 6 response options: less than 1 week; 1 week up to 2 weeks; more than 2 weeks up to 4 weeks; more than 1 month up to 3 months; more than 3 months up to 6 months; or more than 6 months up to 12 months. We aggregated response categories as more than 2 weeks, more than 1 month, or more than 3 months.12The patient interval was defined as the time from symptom onset to first presentation to the general practitioner or accident and emergency based on responses to the structured questionnaire.23 Information on cancer type was extracted from cancer registry data. There were 70 patients (8%) for whom information could not be obtained from the cancer registry; for these patients, cancer site was taken from the self-completed survey response and cross-validated against the extracted clinical information from case report forms.Gender, age at diagnosis, and residential postal code , were extracted from case report forms, whereas information on ethnicity, relationship status, and employment status was elicited through the survey via self-report.24 for cancers common among the adolescent and young adult population according to their diagnoses as coded by the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision. The 9 categories were as follows: leukemia; lymphoma; central nervous system and other intracranial and intraspinal neoplasms ; osseous and chondromatous neoplasms, Ewing tumor, and other neoplasms of bone (bone tumors); soft tissue sarcomas; germ cell and trophoblastic neoplasms (germ cell cancers); melanoma and skin carcinoma; carcinomas (except of the skin); and miscellaneous specified neoplasms not elsewhere classified and unspecified malignant neoplasms not elsewhere classified (grouped as unclassified or unspecified).Cancers were grouped into 9 categories using the morphology-based classification of Birch et al12We described the proportion of patients with a patient interval of more than 1 month by presenting symptom, stratified by cancer site  associated with each cancer group. Subsequently, we described proportions of patients with longer patient intervals by presenting symptom using the aforementioned 3 aggregated categories: more than 2 weeks, more than 1 month, and more than 3 months. All analyses were conducted using STATA SE statistical software version 15.1 (StataCorp). Data analysis was performed from January 2018 to August 2019.12 Lump or swelling was the most common symptom , followed by extreme tiredness  and unexplained pain . Night sweats (192 patients), lymphadenopathy (191 patients), and weight loss (190 patients) each were reported by almost one-quarter of patients before diagnosis . Seven of the 17 symptoms were reported by less than 10% of the study population  were male, 509 (63%) were aged 19 to 24 years, and 705 (88%) described their ethnicity as White, as detailed previously.pulation .Overall, there was substantial patient-level variation in symptoms experienced before cancer diagnosis, with 557 patients (69%) reporting multiple symptoms. Patients with leukemia (88 patients [86%]) and lymphoma (200 patients [77%]) were most likely to report multiple symptoms, whereas those with melanoma were least likely to report multiple symptoms (9 patients [31%]) . In totaThe symptom signature of presenting symptoms varied by cancer group . Some syExtreme tiredness was 1 of the 5 most frequent symptoms experienced before diagnosis among 8 of 9 cancer groups, ranging from 10 patients with bone tumors (13%) to 74 patients (73%) with leukemia. Night sweats was 1 of the 10 most frequent symptoms listed for all 9 cancer groups.Almost one-half (385 patients [48%]) of the 745 patients with complete information on the patient interval had a patient interval longer than 2 weeks, 204 patients (27%) had an interval longer than 1 month, and 91 patients (12%) longer than 3 months. The proportion of patients with patient intervals longer than 1 month varied by presenting symptom, ranging from 6% (1 patient) for fits or seizures to 43% (18 patients) for recurrent infections, with variation by cancer site  of adolescent and young adults with cancer presented more than 1 month after symptom onset, with some observed variation between presenting symptoms.26 Most existing studies27 on adolescents and young adults are based on information captured in EHRs. In comparison, our findings based on self-reported data indicate substantially higher frequencies of presenting symptoms among adolescent and young adults before cancer diagnosis. This is in line with previously observed disparities between self-reported and EHR-based symptom frequencies among adult populations.30Literature on the nature and frequency of symptoms experienced by adolescents and young adults before cancer diagnosis is sparse; study populations often exclude those older than 16 years.26 that examined 3 categories of symptoms  among primarily older children and adolescents  with solid tumors found no statistically significant evidence for variation in time to help-seeking. In our study, the sample size precluded the use of multivariable regression to examine differences in patient interval between individual symptoms while adjusting for potential confounders.31 Nonetheless, our observed (crude) findings are compatible with those reported by Veneroni and colleagues26; namely, the proportion of patients with intervals longer than a month was broadly similar between different presenting symptoms.Regarding associations between symptoms and diagnostic timeliness, a previous studyOur findings show that adolescents and young adults with cancer present with a higher frequency of symptoms than previously estimated. Furthermore, although some cancer types have relatively distinct symptom signatures dominated by a single symptom , many presenting symptoms are shared across multiple cancer types and are often vague and nonspecific in nature, such as extreme tiredness, unexplained pain, and night sweats.Understanding the presenting symptoms of adolescents and young adults with cancer could inform the design of interventions to raise awareness of cancer symptoms. Although we described variation in the timeliness of help-seeking among the 17 studied symptoms, future (larger) studies could examine this accounting for patient characteristics and cancer group.19 For example, lump or mass has previously been reported as being present in 24% of patients with lymphoma,19 whereas patient-reported data in the present study indicate a frequency of more than 50%; similarly, fatigue has been reported as being present in 11% of patients with leukemia19 compared with a frequency of 73% in our data. These observations suggest that substantial underestimation of symptoms among adolescents and young adults with cancer is likely when using information from structured fields in EHRs, because of incompletely coded entries or incomplete reporting of symptoms to the general practitioner by adolescents and young adults.Our results indicate substantially higher prevalence of presenting symptoms among adolescents and young adults with cancer compared with previous research based on structured items (coded entries) in EHR data.19 If the degree of undercoding of symptoms is differential between cases and controls, then this would bias the estimation of the PPV. Specifically, greater undercoding in cases more than controls would affect the true-positive and false-negative counts, and lead to underestimation of the PPV. In comparison, greater undercoding in controls more than cases would affect false-positive and true-negative counts, leading to overestimation of the PPV. Further work examining the prevalence of presenting symptoms among adolescents and young adults with and without cancer is, therefore, merited. Use of optimized EHRs, including free-text information ,32 or combining both EHR-derived information and self-reported data may be informative.34It is worth considering the potential implications of the underestimation of true symptom frequency on previously reported positive predictive values (PPVs) of presenting symptoms.22To our knowledge, this is the largest study to date to examine the self-reported symptoms of adolescents and young adults before cancer diagnosis. Our findings are based on a large cohort of adolescent and young adult patients with cancer, encompassing self-reported data on both symptoms and timeliness of help-seeking. Survey participants were largely representative of incident cancer cases, with some underrepresentation of brain tumors, melanoma, and carcinomas.31Although the BRIGHTLIGHT cohort represents one of the largest adolescent and young adult study populations with diagnostic pathway information, the broad spectrum of presenting symptoms in the cohort meant that analyses adjusting for potential confounders of interest, such as patient characteristics and cancer group, were not possible. Specifically, the number of patients with the outcome of interest  relative to the number of exposures of interest (17 symptom categories) was too small for adjusted analyses.35Self-reported information on symptoms and intervals to help-seeking collected retrospectively is associated with risk of recall bias, and a priori excludes patients with poor prognosis who may die soon after their diagnosis, leading to survival bias. We observed much higher estimates of symptom prevalence before diagnosis than those previously estimated using data from EHRs, which are prone to underrecording of symptoms and their duration because of incomplete disclosure or elicitation during the consultation, and/or incomplete entering of information in the patient’s EHR, particularly if only coded entries are used for research.Adolescents and young adults with cancer present with a broad spectrum of symptoms, some of which are common across different cancer groups. The degree of observed variation in time to help-seeking by presenting symptom is not large enough to enable specific targeting within awareness campaigns. Our findings indicate substantially greater frequencies of presenting symptoms among adolescents and young adults with cancer than previously reported; re-evaluation of their prevalence and PPV is warranted.


In this review, I highlight unique features of early life immunity and explore the role of intestinal bacteria in educating the developing immune system.The early life immune system is characterized by unique developmental milestones. Functionally diverse immune cells arise from distinct waves of hematopoietic stem cells, a phenomenon referred to as ‘layered’ immunity. This stratified development of immune cells extends to lineages of both innate and adaptive cells. The defined time window for the development of these immune cells lends itself to the influence of specific exposures typical of the early life period. The perinatal immune system develops in a relatively sterile fetal environment but emerges into one filled with a multitude of antigenic encounters. A major burden of this comes in the form of the microbiota that is being newly established at mucosal surfaces of the newborn. Accumulating evidence suggests that early life microbial exposures, including those arising  The bottom line is that many infectious diseases affect children differently than adults. Understanding these differences, including how the immune system develops and the environmental and host factors that dictate plasticity in early life immune responses, will yield important insight into disease pathogenesis and inform development of new therapeutics.At the time of writing this review, the world was coming to grips with the scale of the COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2. The virus had infected millions of people worldwide and claimed hundreds of thousands of lives. Epidemiological data indicated, however, that the disease affected far fewer children than adults. Children and infants that did test positive for SARS-CoV-2 were largely asymptomatic or experienced a mild form of the disease. Children are usually more susceptible to respiratory viral infections and not less prone to developing severe acute respiratory distress syndrome (ARDS), a grim presentation of COVID-19. As demonstrated in the H1N1 flu pandemic of 2009, being under the age of 1 year was a significant risk factor for ARDS and the more severe form of the disease.6 Distinct immune cell subsets arise in early life that are wired to function in an environment with unique exposures. One exposure of critical importance is the burgeoning intestinal microbiota, the collection of diverse bacteria, archaea, viruses, and fungi, that regulate aspects of immune system development and function. Here, I review the premise for an early life time window when microbial encounters influence the development of select immune phenotypes.The early life represents a period of unique immune development during which the foundation for lifelong immunity is laid. Epidemiological data link pre- and post-natal encounters with susceptibility to chronic inflammatory and metabolic diseases in later life.7 The developing fetus may respond to hostile uterine environments, resulting, for example, from poor maternal nutrition or infection, by changing endocrine and metabolic activity that slow down its growth rate. Resulting irreversible changes in the structure and function of vital organs are exacerbated by subsequent environmental exposures during infancy and childhood that condition the risk of disease in later life. A critical foundation of the DOHaD hypothesis is the concept of developmental plasticity, or the ability of an organism to develop in diverse ways, depending on the particular environment and setting.8 The immune system displays considerable developmental plasticity. Hematopoietic stem cells (HSCs) that give rise to numerous blood and immune cells are able to sense diverse stimuli to influence the type and function of cell lineages generated.11 Plasticity is also observed during the generation of antigen-specific immune responses, the quality of which are determined by numerous macro- and micro-environmental factors that regulate the differentiation of specific effector cells.13 Thus, the principles of the DOHaD hypothesis are applicable to the immune system too, and numerous studies have established links between maternal nutritional or infectious status and the offspring’s immunity and subsequent disease risk.15 For example, maternal malnutrition results in decreased placental transfer of maternal antibody to the fetus. This has long-lasting effects on B cell development in offspring that correlates with susceptibility to allergies and autoimmune diseases.17 Chronic helminthiasis during pregnancy promotes Th2 cytokine responses and regulatory T cell differentiation in offspring19 that correlates with diminished vaccine responses and increased susceptibility to tuberculosis.20The Developmental Origins of Health and Disease (DOHaD) hypothesis proposes that exposure to certain environmental stimuli during critical periods of prenatal development have significant consequences on an individual’s short- and long-term health.21 Shared living spaces and air was presumed to increase microbial spread in younger children of large families, which favored the development of protective immunity to hay fever in younger siblings. Similar observations have since been made for a variety of environmental conditions that promote microbial exposure early in life. The prevalence of atopy was found to be lower in children who started to attend day care at a younger age (6–11 months) compared to those who started at an older age.23 Farm living is associated with exposure to higher diversity of bacteria and fungi that appear to protect from atopy.24 Conversely, higher incidences of asthma and hypersensitivity disorders were observed in children raised in ‘cleaner’ urban dwellings compared to rural and farm environments.27 Thus, while the DOHaD hypothesis proposes a prenatal developmental period during which adversarial exposures increase the risk of disease, the ‘early life window of opportunity’ argument advances the notion of a defined perinatal period during which specific microbial exposures lead to a favorable imprint on immunity that reduces risk of disease later in life. As windows of opportunity go, it is predicted that this period of immune malleability would be short, would not appear again in later life and would lead to long-term beneficial health consequences. It is of interest, therefore, to understand1 when the window of opportunity arises in early life,2 the identity of immune cells generated during this period that are likely to be receptive to microbial influence,3 the nature of microbes and their components that play a role in educating the early life immune system and of course,4 the practical implications of this phenomenon to ultimately develop strategies that promote health and prevent the incidence of chronic inflammatory diseases. I discuss these in some detail below.However, while unfavorable early life exposures certainly increase the risk of disease in offspring, nonpathogenic microbial exposures may in fact promote the generation of protective immunity. This idea was first proposed by Strachan in 1989 to explain the inverse correlation between birth order and incidence of allergic diseases in families in Britain.in utero during pregnancy, microbes acquired at the time of delivery, and microbes established postnatally by the acquisition of maternal and environmental bacteria , from the mother to the offspring where they primed the developing immune system. Maternal enteric microbes also ferment dietary fibers to the short-chain fatty acid (SCFA) acetate, that suppresses allergic airway disease, a mouse model for human asthma, by enhancing Treg cell numbers and function in adult offspring.40 In the context of a maternal infection, bacterial peptidoglycan, which is a ligand for Toll-like Receptor 2 (TLR2), traversed the placenta to influence fetal neuro-proliferation.41 Passage of other microbial TLR ligands across the placenta that influence fetal immune development remains a formal possibility.The maternal microbiota in the gut and reproductive tract undergoes significant changes during pregnancy that is influenced by a combination of hormonal, metabolic, and immunological factors as well as by maternal diet, supplement intake, and antibiotics use.44 Vaginally delivered newborns display early enteric communities resembling the maternal vaginal and fecal microbiota, a result reflective of their prolonged transit through the birth canal. CS babies bypass contact with maternal mucosal surfaces and their initial microbial exposure consists of maternal skin bacteria and other environmental bacteria.45 The use of antibiotics perinatally by mother-infant dyads significantly impacts the neonatal microbiome and likely contributes to the decreased diversity of the enteric microbiota observed in CS infants.46 Studies have attempted to establish a correlation between mode of delivery to disease susceptibility in later life. CS babies may be at higher risk for developing allergies and asthma compared to vaginally delivered babies.48 A study of a Danish register-based cohort reported CS babies to have increased susceptibility for chronic inflammatory diseases including diabetes, arthritis, inflammatory bowel disease, and celiac disease.49 However, another study found no link between delivery mode and risk for celiac disease.50 Numerous factors accompanying birth may complicate interpretation of such studies, including the use of antibiotics, dominant mode of newborn nutrition immediately after birth and maternal stress and health.51 Further, while it is evident that the mode of delivery affects the quality of pioneer microbes introduced into newborns after birth, whether it also impacts subsequent patterns of colonization in the first 3 months of life remains unclear.52 Irrespective of birth mode, any observed differences in the earliest microbial community structure typically disappear after 6 months of life with the diversification of the infant diet.54Parturition is the next major point of microbial exposure for the developed fetus. The first microbial contacts of a newborn outside of the uterus are influenced by the mode of delivery and the presence of microbial communities on the maternal skin and in the reproductive tract. While the composition of the neonatal microbiota across different body sites early after birth is not impacted by the delivery mode, gut bacterial communities display significant differences in babies born by either vaginal or Cesarean-section (CS) delivery.56 The TEDDY study established that breast milk consumption was the most significant factor associated with the microbiome structure early in life.57 Numerous epidemiologic studies have documented differences in the composition of the gut microbiota in breastfed and formula-fed infants.59 Breastmilk itself contains viable bacteria originating from the maternal gut and infant oral cavity that influences the composition of the enteric microbiome.60 Exclusive breast-feeding in the first 4 months of life was found to prevent atopic dermatitis (AD) in infants at high risk of atopy.61 However, in another study, breast-feeding for at least 3 months was not significantly protective against the development of AD compared with partial breast-feeding or use of conventional formula.62 In addition to nutrition, numerous environmental and host factors also contribute to the step-wise progression of the infant microbiome toward adult-like configurations that are typically reached by 2–3 years of age.63Nutrition, in the form of breastmilk, formula milk, and solid weaning foods, has a defining role in establishing colonization patterns of the infant gut microbiota after initial maternal inoculum at delivery.64 Distinct hematopoietic stem cells give rise to unique lineages of lymphoid and myeloid cells at specified times during development, creating layers of immune cell populations that collectively endow the immune system with vast capabilities  and neonatal sepsis are a significant threat in the early life period.67 This susceptibility tracks with the development of the immune system, occurring within specific time windows that parallel waves of immune cell generation and tissue colonization.68 For example, while immune cells migrate to the skin and gut during gestation, lung resident Th2 effector cells and ILCs are only found after birth,70 contributing to the increased susceptibility to RSV infection in newborns.72 A discussion of the perinatal generation of immune lineages follows to provide a framework for assessing impact of microbial exposures on developmental immune imprinting.The early life mammalian immune system has evolved to develop in functional layers, likely to counter an array of exposures over ontogeny.bilities . Immune Yolk sac and aorta-gonad mesonephros (AGM) hematopoiesis: Immune cells that arise from the first wave of hematopoiesis in the yolk sac have unique structural and physiological functions and equip the embryo with a repertoire of innate effectors consisting primarily of macrophages, mast cells, and Natural Killer (NK) cells. Long-lived macrophages generated from yolk-sac progenitors persist in tissues such as the liver, lung, brain, and epidermis,74 while those from HSC-derived monocytes are found in the gut, lung, and heart.75 Mast cells also display a similar ontogeny to macrophages and may arise from both yolk-sac and HSC-derived precursors that imprint them with distinct patterns of tissue residency.77 Pre-thymic lymphoid progenitors with long-term T cell engraftment capacity are found in the AGM region and arise before the appearance of embryonic HSCs in humans.80 In murine embryos, a subset of innate-like B cells called B-1 cells arise from hematogenic endothelia in both the yolk sac and the AGM region prior to HSCs, and seed the fetal liver.81Fetal liver hematopoiesis: The next wave of hematopoiesis originates from the fetal liver where definitive HSCs with the full complement of erythroid, megakaryocyte, myeloid, and lymphoid potential may be found. Numerous innate and innate-like lymphocytes arise from common lymphoid progenitors in the fetal liver including innate lymphoid cells (ILCs), γδ T cells, and mucosal-associated invariant T cells (MAIT cells).82 While most mature ILC subsets are absent from lymphoid and non-lymphoid tissues such as the skin, kidneys, lungs, and spleen in early fetal development,82 ILC2s and ILC3s are present in the fetal gut.84 Antigen-presenting cells (APCs) including monocytes/macrophages, plasmacytoid dendritic cells (pDC), and conventional DC (cDC) subsets may be found in the fetal spleen, skin, thymus, and lungs by 12 weeks post-conception (PCW).85 HSC-derived innate B-1 cells in mice81 and adaptive B cell (B-2) precursors in humans are found in the fetal liver from 7PCW and mature B cells by 9PCW.82Bone marrow (BM) hematopoiesis: At mid-gestation in humans and just before birth in mice, the BM becomes the primary site of hematopoiesis and the major source of B cells. B cells and follicular DCs form follicles in the fetal spleen,87 enabling antigen presentation to B cells. B cells are also found in the fetal intestine where they display phenotypes of follicular and transitional B cells but not plasma cells.84 While neutrophil progenitors abound in the fetal liver and BM, mature neutrophils are only found in the BM by 14 PCW where their numbers remain extremely low88 until immediately after birth.90Thymus: The human thymus begins to develop by 8PCW and the first T cells appear in the periphery by 12–14 PCW.92 Thymus specific progenitors from the fetal liver and bone marrow seed the thymus and both γδ and αβ T cells including regulatory T (Treg) cells arise in the human fetus.96 In mice, however, except for the Vγ3/Vδ5 TCR expressing γδ T cell population that arises by embryonic day 15 (E15), most γδ and αβ T cell subsets arise in the thymus after birth in the first week of life.97 Memory T cells have also been found in the fetal spleen99 and in tissues including the skin100 and intestine,101 suggesting that complete T cell maturation can occur in utero. A unique feature of naïve T cells arising from fetal HSCs is their increased propensity to differentiate into Treg cells upon antigen encounter, a mechanism that likely establishes tolerance and prevents damaging alloreactivity in utero.103 CD1d-restricted Vα24+ invariant natural killer T (iNKT) cells are found in the early fetal thymus104 but their numbers decline with gestational age such that they are very rare in the neonatal thymus. However, iNKT cells are highly prevalent in the small intestines as well as the lungs, spleen, and mesenteric lymph nodes during the second trimester.105 MR-1 restricted MAIT cells are another class of innate-like T cells that have been found in the human fetal small intestine.106108Early life represents a period of dramatic escalation in antigenic encounters by the relatively naïve and still developing newborn immune system. There is significant overlap of fetal and postnatally derived hematopoietic precursor cells in the period immediately after birth that influence the quality of immune cells generated and the overall functionality of the immune system. It is well established that newborns are highly susceptible to infections by pathogens and commensals, mostly due to insufficient, inappropriate, and biased immune responses. Dampened early life immune responses also necessitate pediatric vaccination strategies to include multi-dose immunization schedules with administration of doses later in infancy to increase immunogenicity and induce protective responses. However, overly robust immune responses are also observed in infants in developed countries, who have become increasingly sensitive to acquiring autoimmune diseases such as Type 1 Diabetes (T1D) as well as allergies and asthma. While the neonate has historically been considered to be immature in its immune characteristics, evidence clearly indicates that it is capable of mounting a range of pro- and anti-inflammatory responses.Innate immunity: Cells of the innate immune system are the first responders to the massive antigenic exposure that follows immediately after birth. The manner in which the innate immune system responds in early life is critical for the initiation and maintenance of host defense throughout life. The immune system recognizes microbial pathogens as well as commensal microbes via pattern-recognition receptors (PRRs) that are expressed on a variety of host cells including innate and adaptive immune cells.109 Appropriate regulation of these receptors is essential to prevent damaging inflammation at mucosal sites during the establishment of the commensal microbiota in early life as well as to generate productive responses against pathogenic viruses and bacteria. Failure of these mechanisms, especially in preterm infants, results in devastating disease such as neonatal sepsis and NEC as well as ineffective responses that hamper viral clearance.68 Decreased expression of TLR4 on monocytes from preterm and very-low birth weight infants and their subsequent lack of cytokine production has been suggested to contribute to susceptibility to infections with Gram-negative bacteria.111 The expression and cellular distribution of most other TLRs are similar between term newborn and adult monocytes.112 However, despite similar basal TLR expression, the functional consequences of TLR engagement in neonates are distinct. TLR stimulation of whole blood from preterm infants results in massive production of IL-10 whereas production of Th17 cell-promoting cytokines IL-6 and IL-23 dominates in term infants.112 pDCs are one of the major sources of Type 1 IFN following TLR triggering during viral infections and can be recruited efficiently to the respiratory tract of infants suffering from acute viral respiratory infections. However, they display dampened responses in RSV infected neonates and are defective in RIG-I dependent type 1 IFN responses.113 Interestingly, monocyte derived DCs as well as pDCs in human cord blood can potently produce IL-12p70 and Type 1 IFNs upon exposure to human CMV, HIV, and HSV.116 Thus, context dependent activation of TLRs on pDCs appears to impact innate immune responses to different viral triggers.Adaptive Immunity: Neonatal adaptive immunity has been historically viewed as being deficient or deviant. Studies demonstrated that exposure to antigen before or during the neonatal period led to the inactivation on absence of T cells capable of mounting an immune response, a deficiency referred to as neonatal tolerance.118 Neonatal T cells were also shown to preferentially produce T helper type 2 (Th2) cytokines IL-4, IL-5, and IL-13 upon stimulation,120 a deviant response that increased susceptibility to infections and allergies during early stages of development.121 Accumulating evidence, however, has led to a revision of this paradigm and it is now appreciated that neonatal T cells are a distinct population of T cells with unique capacities that are well adapted to the perinatal period.122 Neonatal and adult T cells are derived from distinct progenitor cells that imprint in them specific functionalities; for example, a preference for Th2 and Treg cell differentiation in CD4+ T cells and the ability for rapid effector production in CD8+ T cells.124 Distinct gene expression profiles of neonatal T cells observed even prior to stimulation suggest that they may be more responsive upon antigen exposure.128 Neonatal T cells also respond to microbial ligands by virtue of their expression of numerous TLRs including TLR2 and TLR5.131 Thus, neonatal T cells appear wired to establish a state of tolerance to self and allogenic antigens at baseline homeostatic conditions, while simultaneously being poised to mount a rapid effector response upon infectious challenge or injury. B cell responses in the neonatal period are also distinct from adult. Fetal precursor cells preferentially give rise to B1-B cells that persist in the developing spleen at least 4 weeks after birth. These cells recognize self and microbial antigens and secrete low-affinity antibodies with broad specificities.132 The newborn B cell compartment primarily consists of naïve and transitional B cells and few memory cells.134Suppressor cells: Specialized populations of suppressor cells are found in the early life period that control inflammation and also contribute to age-specific susceptibility to a pathogen and disease. IL-10 producing regulatory B cells are prevalent in human neonates and play a role in RSV clearance.135 Myeloid-derived suppressor cells are a heterogeneous population of granulocytic or monocytic cells that suppress innate and adaptive immune responses and play a role in establishing fetal tolerance in utero.137 CD71+ erythroid cells are enriched in the spleen of neonatal mice and in human cord blood and suppress both myeloid cell responses and T cell activation through their production of arginase 2.138Unique forces drive early life immunity, and a microbial imprint of this system during the ‘window of opportunity’ may take the form of programming of either the development of immune cell subsets themselves or their functionality. Favorable microbial exposures could direct hematopoietic progenitors down immune developmental pathways that serve unique early life milieus. An alteration in their homeostasis and ability to precisely differentiate may result in losses in immune lineages, or conversely, may cause an increased persistence of fetal-derived immune cell subsets, both scenarios that would impact long-term immune repertoires. Encounters with microbes and microbial components in early life may also program the function of immune cells, some of which take up long-term residence in tissues, to influence host physiological processes and protect from disease throughout life. When and how microbial inputs are interpreted in these distinct early life environments may alter the nature of developmental immune layering, impact long-term immune function, and drive hypersensitivity.Microbial inputs may originate either from endogenous compounds that are synthesized by microbes such as lipopolysaccharide (LPS) and flagellin or by dietary components that are metabolized by the microbiota, for example, SCFAs produced from dietary fiber. Broad manipulations of the microbiome with antibiotic depletion or germfree rearing have provided important insights into the contributions of the early life microbiome on immune development and later life disease susceptibility. However, microbiome interventions applied maternally can alter both the maternal and offspring microbiomes, rendering it difficult to distinguish gestational from neonatal influences on offspring immune outcomes. Identifying mechanisms by which specific microbes and microbial products affect offspring immune phenotypes may alleviate these concerns and numerous reductionist approaches in animal models have been employed to identify such causal relationships. These primarily involve either starting with an immune phenotype and narrowing down the microbiota to identify specific bacteria that influence the phenotype, or starting with bacteria-derived compounds and metabolites, and seeking to understand their effect on the immune system. A discussion of some of these studies follows.in utero was positively associated with atopic dermatitis, food allergies and very-early onset IBD within the first year of life.140 Similarly, antepartum antibiotic exposure of pregnant C57BL/6 mice and colitis-prone IL-10-deficient mice led to altered intestinal bacterial colonization in offspring and increased susceptibility to DSS colitis in adulthood.142 We have shown that antibiotics exposure of pregnant C57BL/6 dams impacts the development of innate-like lymphocytes in the thymus of offspring that alters susceptibility to experimental colitis in later life.143 TF PLZF expressing thymic cells did not develop efficiently in mice that were treated with broad-spectrum antibiotics in the perinatal period, but mice treated in later life were spared. Microbe-induced altered development of thymic innate and innate-like cells in early life persisted into adulthood and contributed to increased susceptibility to DSS-induced colitis that could be reversed by the transfer of PLZF+ cells from mice that developed with normal microbiota in early life. Exposure of pregnant C57BL/6 mice to a cocktail of antibiotics decreased the numbers of circulating bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells (GMPs) in offspring that was associated with increased susceptibility to E. coli K1 and Klebsiella pneumoniae sepsis.89 The balance between Th17 and Treg cells, both important players in mucosal homeostasis,144 was also affected by antibiotics exposure of murine dams. There were reduced numbers of IL-17 producing cells in the intestinal lamina propria of offspring of antibiotic treated murine dams.89 In the non-obese diabetic (NOD) mouse model, antibiotics treatment of pregnant NOD mice also reduced the numbers of IL-17 producing CD4+ and CD8+ T cells in the mesenteric lymph nodes and Peyer’s patches with a concomitant increase in FOXP3+ Treg cells, that protected offspring from T1D.147 Interestingly, however, vancomycin treatment of C57BL/6 breeders reduced colonic Treg cells in offspring and increased their susceptibility to asthma.148 Impaired anti-viral responses and deficient production of IFNγ by CD8+ T cells were observed in neonatal offspring of antibiotic-treated murine dams.149 However, antibiotic treatment in the neonatal period lead to dysregulated vaccine responses with decreased IgG production and increased IFNγ production by T cells in adult mice.150Epidemiologic and experimental data support a major impact of perinatal antibiotics on numerous aspects of immune system development and function that modulate susceptibility to inflammatory diseases in offspring. Antibiotic exposure 89 In GF mice, an early life defect in differentiation potential and self-maintenance capacity of bone marrow GMPs persisted into adulthood and could be restored partially by re-introduction of commensal microbes.151 GF mice also displayed increased serum IgE levels that were only normalized by conventionalization with normal microbiota before 4 weeks of age.152 Neonatal GF Swiss Webster (SW) mice but not GF C57BL/6 mice had higher numbers of iNKT cells in the colonic lamina propria, that increased their susceptibility in models of oxazolone-induced colitis and ovalbumin-induced allergic asthma.153 Elevations in colonic iNKT cells in GF mice were abrogated by re-introduction of conventional microbes at birth but not in adulthood, revealing an early life period during which the gut microbiota affects intestinal iNKT cell numbers. Early life microbes also regulate intestinal transcriptional programs, and alterations in expression of immune-related genes have been documented in GF mice that were only restored by colonization at birth but not in adulthood.154 Maternal microbes also imprint distinct patterns of gene expression in small intestinal mucosa of offspring in a mouse model of gestation-only microbial colonization.39Similar to offspring of antibiotic treated murine dams, reduction in GMPs and lower plasma G-CSF levels were also observed in neonatal GF mice, suggesting that the immune impairments were due to alterations in the perinatal microbiome rather than confounding off-target effects of antibiotics.155 Isotope-labeling studies show the presence of microbial molecules of maternal origin in the placenta and the developing fetus as well as in breastmilk that drive the development of innate immune system and maturation of the intestinal epithelium in murine offspring.39 Microbial ligands for the aryl hydrocarbon receptor, which is expressed in the placenta, liver, and mucus membranes, promoted the development of intestinal NKp46+ ILC3s in the offspring. Components of bacterial cell wall and capsule are recognized by innate pattern recognition receptors expressed on a plethora of host cells in the intestines. Lipopolysaccharide (LPS) could restore impaired anti-viral CD8+ T cell responses in neonatal offspring of antibiotic-treated murine dams,149 suggesting that endogenous LPS from the neonatal microbiome might be required for proper CD8+ T cell function.156 LPS and microbial ligands for other PRRs have been shown to have diverse effect on central nervous system development and function.157 We showed that capsular polysaccharide A (PSA) from Bacteroides fragilis promoted the development of PLZF+ innate-like lymphocytes in the thymus of neonatal mice in a TLR2 dependent manner.143B. fragilis sphingolipid also inhibited iNKT cell proliferation in the neonatal colon that protected from colitis in later life.158 Flagellin is a ligand for TLR5 that is expressed on intestinal epithelial cells primarily in early life.159 Neonatal flagellin and TLR5 interactions influence the composition of the murine microbiota throughout life and impact susceptibility to metabolic syndrome.160 In mice transgenic for a T cell receptor recognizing Lachnospiracea flagellin, antigen-specific RORγt+ Treg cells were generated in the early life period, specifically during the weaning process, and protected them from severe colitis later in life.161 In another TCR transgenic mouse model, recognition of a Staphylococcus epidermidis antigen to generate antigen-specific Treg cells also occurred during the second week of birth and was necessary to maintain tolerance to this antigen in adulthood.162 There is still a dearth of information on effector microbial products and further research is needed to understand the nature of specific microbial molecules that elicit immune modulatory effects in early life.Endogenous microbial compounds can penetrate into the adult murine host, especially from bacteria in the lower small intestine.165 Maternal obesity in murine dams fed a chronic HFD correlated with restricted expansion of hematopoietic stem and progenitor cells (HSPCs) in the fetal liver of offspring and altered lineage specification that resulted in distinct patterns of reconstitution upon adoptive transfer.166 Children with acute malnutrition also have impaired development of their gut microbiota  expression and enhancing Treg cell generation in a mouse model of pre-eclampsia.173 Retinoic acid (RA) is a Vitamin A metabolite whose synthesis by intestinal epithelial cells is regulated by commensal bacteria.174 Maternal RA was shown to regulate CXCL13 production by murine fetal mesenchymal cells and the differentiation of lymphoid tissue inducer (LTi) cells.175 Vitamin B2/riboflavin metabolites produced by the microbiota before weaning promoted the development of MAIT cells in the murine thymus.176In animal models, maternal consumption of a high-fat diet (HFD) altered the microbiota and the nature of microbial exposures in both pregnant mothers and their offspring.178 A practical approach to prevent such pathologies is to identify components of the microbiota that may be used clinically to ensure healthy imprinting in early life. A combination of probiotics, prebiotics, and immunomodulatory metabolites specific to the early life period may then be designed that promote favorable immune outcomes and decrease the incidence of inflammatory pathologies in later life. These may be administered to mothers during pregnancy, to mother-infant dyads postpartum, or directly to the infant.179 Another strategy is the transfer of mother’s vaginal and fecal microbiota at birth to introduce pioneer bacteria that build a robust microbial community that promotes offspring health. While antibiotic treatment during the neonatal period is causally linked to pathological imprinting, it is difficult to envision how one might avoid these treatments given their critical role in clearing infections in infants. Similarly, consumption of high-fat diets by mothers and weanlings can increase susceptibility to metabolic disorders; however, such high-calorie foods are crucial for meeting nutritional needs in developing countries. One strategy might be to prevent microbial alterations brought about by these treatments and behaviors with dietary prebiotics and probiotics.It is evident that the immune system is amenable to beneficial programming by microbes in the perinatal period. Lack of appropriate microbial exposures, either due to microbial perturbation during pregnancy, antibiotics use in the perinatal period, malnutrition, or exposure to low diversity microbiota can lead to pathological imprinting that contributes to increased susceptibility to asthma, allergies, and other chronic inflammatory conditions.Bifidobacterium and Lactobacillus and their use may prevent antibiotic-induced diarrhea and reduce the risk of NEC in preterm infants.180 However, no beneficial effect of probiotics in acute gastroenteritis in children was found.181Bifidobacterium longum subsp. infantis EVC001 is a gut symbiont that can efficiently utilize human milk oligosaccharides (HMOs) into organic acids that lowers intestinal pH and can decrease markers of enteric inflammation in infants.182 In neonatal mice, colonization with Lactobacillus rhamnosus GG enhanced intestinal functional maturation and IgA production that protected from inflammation in later life.183 Probiotics may modulate the composition of the gut microbiota, enhance epithelial barrier function, and direct antagonism against pathogens. However, the precise mechanism of their action remains to be determined and is likely strain dependent. Prebiotics are substrates that are selectively utilized by host bacteria to confer a health benefit. Dietary fiber that is metabolized to SCFAs  by gut microbes has been shown to influence microbial composition and the generation of immune cell subsets.184 Prebiotics such as short-chain galactooligosaccharides and long-chain fructooligosaccharides are routinely added to infant formula and have been shown to increase the abundance of Bifidobacterium.185 However, further studies are needed to determine their impact on immune function and the composition of the microbiota in early and later life. Crucial information is also needed on the possible long-term effects of early life pro- and pre-biotic administration on immune function and disease susceptibility. Probiotics in infant formula can deliver a large bacterial inoculum into a still-developing intestinal environment. Alterations in intestinal epithelial gene expression profiles, immune cell distribution, and function as well as the microbial landscape could potentially persist into adulthood leading to unwanted long-term health consequences.Commonly administered probiotics belong to the genera −/-Tlr5 mice upon vaccination with a trivalent inactivated influenza vaccine, that was restored by reestablishment of the microbiota.186 Similarly, intestinal microbiota and recognition of the Nod2 sensor were required for inducing an immune response to cholera toxin in mice.187 In humans, variations in microbial communities may provide one explanation for the poor response to oral vaccines in low-income countries. While a causal link between specific community composition and vaccine responsiveness still needs to be established, some bacterial species have emerged as modulators of humoral and cellular immunity in responders. A robust IgA response to oral rotavirus vaccine in Ghanaian infants correlated with an increased relative abundance of stool Streptococcus bovis and a decreased abundance of Bacteroidetes.188 A higher polio-specific T cell response and serum IgG levels in Bangladeshi infants that received oral polio vaccine correlated with a higher abundance of Bifidobacteria and lower Pseudomonadales.189 Approaches to test the immunomodulatory and adjuvant effects of probiotics on beneficial vaccine responses need to be defined.190 As with any probiotic use in early life, choice of strain and dose and timing of administration must carefully be evaluated along with long-term health consequences.Efficacy of vaccine responses can also be potentially programmed by intestinal microbes that may act as immunomodulators as well as natural adjuvants. Low antigen-specific plasma cell frequencies and IgG levels were observed in germfree and antibiotic-treated 177 In mice, a ‘weaning reaction’ encompassing the first 2–4 weeks of life was demonstrated to be critical for mitigating disease risk in adulthood.191 Distinct immune cell subsets are generated from precursor cells arising sequentially from the yolk sac, fetal liver, and bone marrow and may be subject to imprinting by specific exposures arising during these times. Animal models have been vital in providing insights into the perinatal development of the immune system. However, developmental timelines differ between species as do the quality of microbial exposures in the perinatal period. The influence of in utero microbial exposures on human fetal immune development and hematopoiesis remains largely unknown. Most studies have utilized cord blood at the time of parturition to make inferences on the earliest immune cells. However, human cord blood measurements are not representative of postnatal immunity, and display diverse immune phenotypes that appear to converge in the first few weeks of life.52 Further, while cell subsets such as B, NK, and DCs reach adult-like phenotypes during the first 3 months of life, T cell phenotypes do not converge during this period, suggesting that specific cell populations have different periods of calibration and time frames when they are amenable to microbial imprinting.52 Recent single-cell multi-omics technologies utilizing small numbers of cells from human fetal tissue have provided a new-detailed view of the complex immune development and structural and cellular communications occurring over ontogeny.192 More studies are needed to precisely define the temporal contributions of specific microbial species of maternal and neonatal origin on immune cells and their function.In summary, the maternal and neonatal microbiomes are important regulators of health and influence the development of the early life immune system. The precise time frame for this ‘window of opportunity’ when immune cells are likely to be malleable to microbial influences is as yet undetermined. This period likely spans the time from fetal development to weaning or introduction of solid foods . SpecifiReductionist approaches in animal models have been used to delineate causal relationships between early life microbes and disease susceptibility. However, while these studies have been and will continue to be critical for understanding basic mechanisms of early life host-microbiome interactions, how they may be translated to clinical applications and the development of microbiome based therapeutics will be key. Efforts to better model clinically relevant microbiome perturbations will be important as microbiome-based therapeutic strategies are developed. Applying pharmacologically relevant doses of antibiotics to animals, evaluating their off-target effects, and elucidating how microbiome perturbations might impact developmental processes will be important to evaluate safety, efficacy, and promise of such endeavors. Deeper mechanistic and functional insights into microbe-immune interactions at specific time periods are thus critical to inform disease mitigating strategies that modulate early life immune imprinting.


The brain is responsible for maintaining whole-body energy homeostasis by changing energy input and availability. The hypothalamus and dorsal vagal complex (DVC) are the primary sites of metabolic control, able to sense both hormones and nutrients and adapt metabolism accordingly. The mitochondria respond to the level of nutrient availability by fusion or fission to maintain energy homeostasis; however, these processes can be disrupted by metabolic diseases including obesity and type II diabetes (T2D). Mitochondrial dynamics are crucial in the development and maintenance of obesity and T2D, playing a role in the control of glucose homeostasis and whole-body metabolism across neurons and glia in the hypothalamus and DVC. Fission is mediated by the GTPase dynamin-related protein 1 (DRP1) and occurs when mitochondria are damaged or subjected to high levels of cellular stress . They are dynamic organelles that change morphology in response to energy demand and supply D. They ar stress , 4; thisr stress –7. MFN1 c lethal , 8–10, dCircuits within the central nervous system (CNS) control whole-body energy metabolism. The hypothalamus and the dorsal vagal complex (DVC) have been highlighted as crucial regulatory centers of metabolism, and dysfunction of mitochondria in these regions has been implicated in metabolic disorders. The arcuate nucleus (ARC) of the hypothalamus and area postrema (AP) of the DVC are sites with increased blood–brain barrier (BBB) permeability allowing for detection of circulating nutrients, such as glucose and free fatty acids, and hormones that have restricted entry to other brain regions . The abiIn this review, we discuss the contribution of mitochondrial dynamics and dysfunction in the brain to the central control of metabolism and glucose homeostasis and their role in the metabolic disorders obesity and T2D.The body must respond and adapt to changing levels of energy input and availability by altering metabolism. This occurs at the molecular and transcriptional levels to give systems level changes and alterations in behavior. Mitochondria respond to nutrient availability by fusion or fission to maintain homeostasis; however, these processes can be disrupted by metabolic disease. Obesity and its many co-morbidities are associated with chronic inflammation, ER stress, and disrupted mitochondrial dynamics in several tissues [reviewed in –20].β-oxidation, a process where fatty acids are broken down to generate acetyl-CoA for use in the citric acid cycle  using nutrients and oxygen in a series of stepwise reactions that occur at the inner mitochondrial membrane (IMM). This process is the main source of reactive oxygen species (ROS), important for cell functions and acting as a signaling molecule; however, high ROS levels are associated with cell damage. Excessive nutrient intake overloads the mitochondria with glucose and fatty acids leading to an increase in ROS production and associated oxidative stress in the skeletal muscle , 22. Obeid cycle . The redcess ROS . High-facess ROS . When ROcess ROS . ROS act tissues  and acti tissues , 29. Lev tissues , 31, and tissues , illustr tissues . Immune in vitro study found that ER stress and the UPR cause insulin resistance by depleting the cell surface population of the insulin receptor, impairing the transport of newly synthesized proreceptors (ob/ob) before the development of obesity at postnatal day 10 (P10) (The endoplasmic reticulum (ER), a complex protein folding and trafficking organelle, interacts with the mitochondria and is another source of ROS. The ER can transduce signals from lipid metabolites and cytokines to stress kinases and plays an important role in insulin resistance . ER-medieceptors . This ef10 (P10) . Adminis10 (P10) . ER streAlong with an upregulation in mitochondrial ROS production, dysfunction in mitochondrial dynamics is also associated with metabolic disease. A decrease in number of mitochondria and reduction in bioenergetic capacity have been measured in T2D patients . ReducedClear roles for aberrant mitochondrial dynamics have therefore been shown in peripheral tissues during the development of insulin resistance and obesity. Oxidative stress and ER stress are associated processes that both result in inflammation and are linked to the development of insulin resistance in many tissues including liver, skeletal muscle, adipose tissue, and brain. Mitochondria are crucial in maintaining energy balance and are therefore key players in the pathophysiology of metabolic disorders in peripheral organs as well as in the central nervous system.via PI3K/Akt pathway and subsequent ATP-sensitive K+ (KATP) channel activation, alleviating the inhibitory constraints on POMC neurons  is critical for the regulation of appetite, feeding behavior, and body weight. Specific populations of neurons within the ARC respond to circulating hormones including insulin, leptin, and ghrelin to maintain energy homeostasis . AgRP/NP neurons , 54. Inshe liver  and promhe liver . Leptin he liver –59. The he liver , and bothe liver .via MFN1 and MFN2  caused leptin resistance, hyperphagia, and a reduction in energy expenditure and obesity  in the DVC of healthy regular chow fed rats, insulin resistance was induced  prevented changes to mitochondrial dynamics induced by HFD , which transport glucose across the plasma membrane by a facilitated diffusion mechanism , and dorsomedial hypothalamus (DMH)  Figure–108.ATP channels which couple the energy status of the cell and electrical activity, closing the channel and triggering depolarization in a high ATP/ADP ratio and opening the channel causing hyperpolarization in a low ATP/ADP ratio  caused a loss insulin signaling activation of Akt in astrocytes with no effect on peripheral insulin signaling. Astrocyte-specific loss of insulin receptors decreased hypothalamic glucose sensing as GFAP-IR-KO blunted the activation of POMC neurons to cause an increase in systemic glucose levels , and altered glucose metabolism  which increases neuronal firing  pump has been evidenced as one of the molecules which converts low glucose levels to changes in neuronal excitability in glucose sensitive cells  diet for 3 weeks . HFHS-feUcp2 KO mice  to monitor changes in glucose availability and mediate cellular utilization of glucose, GCK therefore links increased glucose to changes in membrane potential which occur via glucokinase-dependent modulation of KATP channels  [reviewed in –188]. InWhile we have some understanding of how mitochondrial dynamics in the brain are associated with food intake and glucose sensing, there are still many more questions to be answered. The cell types involved in the hypothalamus have begun to be dissected, but as yet there is little data on the roles of specific cells in the DVC, and indeed for both regions the role of mitochondria in astrocytes and microglia needs to be further explored. Mitofusins and OPA1 also need to be studied in the DVC in relation to overnutrition, obesity, and T2D. This review has focused on diet-induced obesity and T2D; the genetic etiology of obesity remains unknown, but genome wide association studies (GWAS) of T2D have identified variants associated with T2D . Future A fine balance of mitochondrial network dynamics is required for healthy brain function, T2D and obesity are modifiable risk factors that if ameliorated could reduce the risk of developing neurodegenerative comorbidities, and targeting mitochondrial dysfunction in the brain could be the link between these disorders. It is clear that mitochondria are crucial in the development and maintenance of obesity and T2D, playing a role in the control of glucose homeostasis and whole-body metabolism across cell types in the hypothalamus and DVC.JH and LN contributed to the initial concepts and wrote the manuscript. BF contributed to the initial concepts and revised the manuscript. All authors contributed to the article and approved the submitted version.Our lab is funded by: Royal Society (RG160605), Diabetes UK (2338) and Wellcome Trust (UNS63234). BF is supported an MRC-Career Development Fellowship (MR/S007288/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The past decade has seen several critical advances in our understanding of hypothalamic–pituitary–adrenal (HPA) axis regulation. Homeostatic physiological circuits need to integrate multiple internal and external stimuli and provide a dynamic output appropriate for the response parameters of their target tissues. The HPA axis is an example of such a homeostatic system. Recent studies have shown that circadian rhythmicity of the major output of this system—the adrenal glucocorticoid hormones corticosterone in rodent and predominately cortisol in man—comprises varying amplitude pulses that exist due to a subhypothalamic pulse generator. Oscillating endogenous glucocorticoid signals interact with regulatory systems within individual parts of the axis including the adrenal gland itself, where a regulatory network can further modify the pulsatile release of hormone. The HPA axis output is in the form of a dynamic oscillating glucocorticoid signal that needs to be decoded at the cellular level. If the pulsatile signal is abolished by the administration of a long-acting synthetic glucocorticoid, the resulting disruption in physiological regulation has the potential to negatively impact many glucocorticoid-dependent bodily systems. Even subtle alterations to the dynamics of the system, during chronic stress or certain disease states, can potentially result in changes in functional output of multiple cells and tissues throughout the body, altering metabolic processes, behavior, affective state, and cognitive function in susceptible individuals. The recent development of a novel chronotherapy, which can deliver both circadian and ultradian patterns, provides great promise for patients on glucocorticoid treatment.  Pulsatile glucocorticoid production arises due to a subhypothalamic pulse generator and is the intrinsic property of the feed-forward feedback interplay between the pituitary and adrenal glandsThe pulsatile hormone signal is decoded at the cellular level by the intracellular glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), or both GR and MR in cell types where the 2 are coexpressedLong-standing models of GR and MR working in collaboration and in opposition have retained their validity for the most part, with recent evidence for a role of MR in increasing GR transactivation potential during pulsatile glucocorticoid treatment, via a tethering mechanismPulsatile glucocorticoids have now been demonstrated to be required for optimal HPA physiological responses, stress-coping behavior, complex cognitive processing, glutamatergic neurotransmission, synaptic metaplasticity, and emotional processing in man and experimental rodentsAs glucocorticoid ultradian dynamics are altered in myriad disease states, as well as during synthetic glucocorticoid treatment and glucocorticoid replacement therapy, the resulting effects of GR- and MR-expressing cells and tissues can induce detrimental effects on physiological, cognitive, and behavioral functionStrategies to normalize circadian and ultradian endogenous glucocorticoid rhythms—along with more refined chronotherapies that are able to integrate both circadian and ultradian rhythms into their design—are currently being developed and show great promisesensitive to environmental perturbations, and to be able to respond differentially to small and large stimuli. This responsiveness must be (3) robust with preservation of dynamic behavior during these perturbations. Finally the system must show (4) plasticity to facilitate adaptation to new circumstances. This concept of dynamic regulation in endocrinology refines the older concept of homeostasis toward a steady-state set point to a more dynamic understanding of how systems oscillate around an equilibrium position and how this allows for a reactive and adaptive system. Furthermore, it provides a conceptual basis for how allostasis—a new dynamic equilibrium position in response to novel circumstances—can lead to physiological change and disease. With these thoughts in mind our review will bring together new concepts of continuous dynamic equilibration, and how these provide the basis for understanding the importance of both circadian and ultradian rhythmicity for a responsive and adaptive HPA axis.The hypothalamic–pituitary–adrenal (HPA) axis is a neurohormonal system that is critical for life. It is a multisystem axis that utilizes feed-forward and feedback loops to regulate glucocorticoid hormone levels within the physiological range appropriate for system homeostasis. This is an equilibrium control system we have called continuous dynamic equilibration . This syWithin the hypothalamus, the parvocellular neurons of the paraventricular nucleus (PVN) are a group of densely packed neurons that are highly responsive to external physiological stimuli such as altered light/dark cycle, or the presence of real or perceived stress ,3, as shzona fasciculata of the adrenal cortex to activate the synthesis and subsequent release of glucocorticoid hormones , significant differences were found in the increased fasting serum cortisol  as well as in the increased frequency of the cortisol pulses   . As huma< .0001) ,50, therThere are very clear similarities in the regulation of HPA dynamics found in experimental rodent studies and clinical studies in man. Consistent with the dynamics of the pituitary adrenal system observed in man , each puThe general notion of a hypothalamic pulse generator prevailed until quite recently , despiteAlthough the pituitary–adrenal interaction is the mechanism underlying ultradian rhythmicity, hypothalamic modulation of the HPA axis is the dominant factor regulating pulse amplitude over the course of each day. Circadian rhythms in activity, body temperature, and hormonal systems are tightly controlled by the central circadian pacemaker, the suprachiasmatic nucleus (SCN) in the hypothalamus, which receives light cues via the retinal projections to entrain to a 24-hour rhythm ,60. ConsOne of the key properties of the pituitary adrenal interaction is the inherent delay in ACTH-induced glucocorticoid release from the zona fasciculata of the adrenal cortex. This is because glucocorticoid hormones, lipophilic molecules, cannot be stored in vesicles but require de novo synthesis by steroidogenesis. For each pulsatile secretory event, steroidogenesis is initiated by ACTH binding to the cell surface G-protein coupled melanocortin type-2 receptor (MC2R) , which aMC2R, StAR, and CYP11A1 (the gene encoding cytochrome P450 side chain cleavage enzyme) to presumably replenish the cellular store of steroidogenic pathway components.Spiga et al. have elucidated a complex regulatory network, which acts together with cAMP response-element binding protein (CREB), notably including the positive regulators SF-1 and Nur77, and the negative regulator Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) as shown in There is also an inherent short delay in the negative feedback part of the loop. Once synthesized, glucocorticoids rapidly travel through the circulation to inhibit the HPA axis at the level of the hypothalamus and anterior pituitary, inhibiting synthesis and release of CRH  and ACTHNR3C2 gene  were both cloned by 1987 ,82 and wThe endogenous glucocorticoids corticosterone and cortisol bind to MR with a 10-fold greater affinity than to GR . During 1/2 = 5 seconds), indicating that each GR binding event at the chromatin template occurred for 10 seconds to 20 seconds before exchanging. While GR’s recruitment was deemed to be highly stochastic, its ejection from the chromatin template was found to be dependent upon ATP and remodeling complexes  is characterized by excess levels of circulating glucocorticoid, most commonly due to exogenous treatment, which will be discussed in detail further into this review. More rarely, but posing a clinically important problem, CS is caused by unregulated excessive production of ACTH by a pituitary tumor (Cushing’s disease) ,143, by Along with myriad metabolic adverse effects and cardiovascular risks , patientObstructive sleep apnea (OSA) is a condition associated with increased prevalence of cardiovascular disease, metabolic syndrome and glucose intolerance , all symAccording to the corticosteroid receptor hypothesis of depression  changes In healthy individuals, adrenal glucocorticoids exert anti-inflammatory and immune modulating actions . The acuAn excellent example of the role of plasticity within the HPA axis is the response to coronary artery bypass graft surgery. Gibbison et al.  used perThis highly advantageous adaptive process of immune modulation of adrenal glucocorticoid secretion  also unfSynthetic glucocorticoids are one of the most commonly prescribed classes of drugs, primarily for treating inflammatory conditions, from skin conditions such as dermatitis to rheumatological diseases such as systemic lupus erythematosus, Crohn’s disease, ulcerative colitis, rheumatoid arthritis and asthma. In the UK and US, nearly 2% of the adult population use oral glucocorticoids at any given time ,202. TrePrior to the development of steroid replacement therapy, adrenal insufficiency resulted in 10% mortality rates . GlucocoWe are now at a critical point for the development of safer and more effective glucocorticoid replacement therapy. One way forward is the development of physiological pulsatile replacement therapy using miniature nanopumps or subcutaneous reservoirs. We have shown proof of principle that subcutaneous infusions can reproduce normal circadian and ultradian rhythmicity . FurtherIt is not only replacement therapy that might benefit from ultradian chronotherapy. Glucocorticoid hormones are currently prescribed to over 1% of the population  and side


AR). It is organized in eight exons located on the X chromosome. Hundreds of genetic variants in the AR gene have been reported in CAIS. They are distributed throughout the gene with a preponderance located in the ligand-binding domain. CAIS mainly presents as primary amenorrhea in an adolescent female or as a bilateral inguinal/labial hernia containing testes in prepubertal children. Some issues regarding the management of females with CAIS remain poorly standardized . Basic research will lead to the consideration of new issues to improve long-term well-being . An expert multidisciplinary approach is mandatory to increase the long-term quality of life of women with CAIS.Complete androgen insensitivity syndrome (CAIS) is due to complete resistance to the action of androgens, determining a female phenotype in persons with a 46,XY karyotype and functioning testes. CAIS is caused by inactivating mutations in the androgen receptor gene ( Androgen insensitivity syndrome  is a main disorder (or difference) of sex development (DSD) with a 46,XY karyotype . AIS is In this paper, some aspects related to the molecular genetics, diagnosis and management of complete AIS (CAIS) are summarized, and some controversial aspects are discussed, taking into account some findings from basic research.The androgen receptor (AR) protein belongs to the superfamily of nuclear receptors also designated as NR3C4 . The AR protein consists of a 920 amino acid sequence, has a molecular mass of 110 kDa and is organized in eight exons (indicated as A–H or 1–8) and seven introns. The AR receptor is a single-stranded polypeptide consisting of four main structural domains . Co. CoAR muAR mutations (about two-thirds) are of germline origin inherited from asymptomatic mothers; in other cases, CAIS is due to somatic and de novo mutations [AR gene, but in the presence of the phenotype as well as biochemical data suggesting AIS, an altered signaling pathway due to the impairment of some coactivators or some postligand binding factor has been suggested. However, mutations in cofactor genes have not been found in the large majority of such cases; if so, this hypothesis will require more investigations [The majority of utations . It has igations .Females with CAIS present with a normal external female phenotype in girls and women with a 46,XY karyotype and normally functioning testes ,2,3,8,9.Today, the diagnosis of CAIS can also arise because of a mismatch between the prenatal sex  and the phenotype at fetal ultrasound scans or at birth. Some series reported this clinical presentation in about 3% , while iIn consideration of the X-linked transmission, it is recommended to perform a karyotype in prepubertal female relatives of a proband, given the possible recurrence of CAIS within the same family .A delay between the initial clinical suspicion and the definitive diagnosis is still present despite advances in molecular techniques in our and other’s experience ,6. DelayMalignant transformation of the gonads is the most feared complication in women with CAIS ,8,9; timGonadectomy has been performed prior to definitive molecular diagnosis in some females, and early gonadal removal may still occur in prepubertal girls ,17,18. HPostponing gonadectomy until after the age of majority requires an accurate follow-up. Döhnert et al.  proposedHypergonadotropic hypogonadism is obviously present in adolescent and adult females with CAIS and removed gonads ,2,3. AdeThere is no unique HRT protocol or any evidence-based data on the optimal hormone formulations, route of administration, doses and monitoring parameters in women with CAIS. Various formulations of estrogens are used to induce puberty or adult HRT. The more adequate formulations are likely oral micronized or transdermal (gel or patch) bioidentical 17-β-estradiol ,26. The Because testosterone is the main steroid hormone secreted by testes in women with CAIS, it may be an alternative therapy to estrogens for these females ,26. ReceProgestins are not indicated due to the absence of a uterus and possible negative effects on bone mineral density (BMD) , but sevInternational surveys should be performed to give evidence-based indications for optimal HRT in women with CAIS and removed gonads. Specific hormonal profiles of these women should be considered when monitoring HRT . Since tAndrogens are involved in the growth, development, peak achievement and maintenance of bone mass . Thus, aAR expression in cultured osteoblasts, which was later confirmed both in vivo and in vitro in osteoblasts and osteocytes. Other authors underlined the role of androgen-enhanced tissue-nonspecific alkaline phosphatase expression mediated by AR for the initial formation of hydroxyapatite crystals during osteoblastic mineralization. AR was found to preserve the number of trabeculae independently from GH/IGF1 effects, acting directly on osteocytes or indirectly by inhibiting osteoclastogenesis through interaction with osteoblast precursors, but it seems to have no direct role on osteoclasts [Experimental data in AR knockout (ARKO) mice demonstrated a phenotype with reduced bone mass and decreased stiffness. Microstructural analysis revealed a significant loss of bone volume and trabecular number with a shift towards bone resorption . In 1989eoclasts ,31. GonaFew data are available on bone health in CAIS , due to AR gene (Some studies have shown reduced BMD in women with CAIS according to both female and male reference values . Study sAR gene  with theThe effectiveness of HRT in normalizing BMD values in gonadectomized women with CAIS remains an unsolved issue . RecentlDespite the administration and good adherence to classic HRT, many women with CAIS reported a decrease in psychological well-being and sexual functioning after gonadectomy ( and persFurther longitudinal randomized trials are needed to identify the advantages of testosterone replacement therapy with respect to classical estrogen therapy in terms of psychological and sexual well-being in women with CAIS.Recent studies of ARKO mouse models uncovered new cell-type- or tissue-specific actions of AR .A decrease in myelocytes/metamyelocytes and mature neutrophils were found in bone marrow cells of ARKO mice. The defect in granulopoiesis occurs during the transition between the proliferation of precursors and the maturation of neutrophils; such defects together with a high rate of apoptosis lead to increased susceptibility to acute bacterial infection . ImpaireImpaired AR signaling can be responsible for an increased risk of metabolic syndrome, diabetes and cardiovascular diseases. ARKO mice became obese with increased body white adipose tissue mass without an increase in food uptake or dyslipidemia at ages beyond 10 weeks old; hyperinsulinemia, hyperleptinemia, hyperglycemia, hypoadiponectinemia, increased serum levels of triglycerides and free fatty acids and impaired glucose tolerance occurred with aging . These dThe ARKO mice model presents with a decreased heart size, smaller volume and wall thickness of the left ventricle and significantly reduced aortic NO synthase expression. Taken together, these observations seem to indicate that AR disruption may play some role in promoting atherosclerosis. It also seems that the AR might play some role in the initiation or progression of abdominal aorta aneurism .Data on these items are largely absent for humans with CAIS. Some of our previous data in a small group of women with CAIS and intact or removed gonads suggested that altered AR signaling may increase body fat and affect some metabolic parameters , but theAR plays key roles during male sex differentiation in utero and produces pleiotropic effects during extrauterine life; the understanding of these roles and effects has been expanded by recent basic researches ,2,3,50.