DUOX2 and DUOXA2, and environmental factors, for example, iodine deficiency and excess, anti- TSHR antibodies and exposure to antithyroid or iodine-rich medications, may cause TCH. Resolution of TCH in childhood may reflect both normal thyroid physiology  and clearance or cessation of environmental precipitants. The relative contributions and interactions of genetic and environmental factors to TCH, and the extent to which TCH may be prevented, require evaluation in future population-based studies.Transient congenital hypothyroidism (TCH) refers to congenital hypothyroidism which spontaneously resolves in the first few months or years of life. Currently, there is a paucity of reliable markers predicting TCH at diagnosis, and the diagnosis is established following the withdrawal of levothyroxine therapy around 3 years of age. The incidence of TCH is increasing, and it is a major contributor to the overall increase in the incidence of CH in recent studies. Both genetic factors, in particular mutations affecting  Nadia Schoenmakers, PhD is a Principal Investigator and Wellcome Trust Senior Clinical Fellow at NIHR Cambridge Biomedical Research Centre, University of Cambridge, UK. Dr Schoenmakers’ principal research interest lies in elucidating the genetic and environmental determinants of congenital hypothyroidism (CH). Her research uses candidate genes, gene panel and whole exome sequencing technologies to identify known and novel genetic causes of CH. She then undertakes phenotyping of genetically ascertained individuals with parallel molecular and murine studies, aiming to gain new insights into thyroid biology and associated extra-thyroidal phenotypes.in situ (GIS CH). Individuals with GIS CH may have a specific disruption of the thyroid hormone molecular biosynthetic pathway (dyshormonogenesis), which is usually genetically mediated and may result in goitre. Primary CH usually results in permanent thyroid dysfunction. However, a sizeable number of affected children will recover endogenous thyroid function in early childhood, permitting the cessation of levothyroxine treatment by the end of the third year of life. This group of children are said to have ‘transient congenital hypothyroidism’ (TCH) and usually exhibit GIS CH. Although some cases of mild dysgenesis and thyroid ectopia may have residual thyroid function, it is uncommon to see cases of TCH in this context  is the most common neonatal endocrine disorder and may be sub-classified as a failure of normal gland development  or failure of thyroid hormonogenesis despite the presence of a normally located thyroid gland  context , 2.This review will clarify the definition and diagnosis of TCH and summarize key knowledge regarding its epidemiology. The process of thyroid hormone biosynthesis will be explained including the roles of known genetic and environmental determinants of TCH in normal thyroid physiology, and the likely contribution of these factors to the pathogenesis of TCH, when perturbed. Despite the prognostic importance of TCH and its increasing frequency, much remains unclear regarding its pathophysiology. These uncertainties, and areas mandating further study, will be delineated.The introduction of newborn screening for CH has been a major public health success, enabling early commencement of levothyroxine in affected infants and thereby significantly ameliorating CH-associated physical and neurodevelopmental morbidity. Following the detection of elevated thyroid-stimulating hormone (TSH) or subnormal thyroxine (T4) levels , the diagnosis of CH is substantiated by venous thyroid function tests. A European consensus has determined that a raised TSH level of greater than 20 mU/L and free T4 (FT4) concentration below the age-appropriate reference range confirm a diagnosis of primary CH and mandate prompt treatment with levothyroxine . ThyroidForty years ago, the majority of CH cases were attributed to thyroid dysgenesis. Over time, the incidence of CH has increased worldwide; key recent epidemiological studies are summarized in In some regions, the increased incidence of CH is largely due to increased detection following the lowering of screening thresholds or altered screening algorithms , 7, 8, 9A smaller number of studies have specifically evaluated the proportion of CH attributable to TCH, with most demonstrating an increase over time, such that this may now include more than one-third of children with GIS CH , 7, 16. Circulating thyroid hormone concentrations in euthyroid individuals are at their highest in the neonatal period but decrease rapidly during infancy, reflecting a significant decline in thyroid hormone requirements during the first year of life . SubsequHowever, GIS CH includes a spectrum of variably severe hypothyroidism, in which the magnitude of thyroid dysfunction will be indirectly proportional to the efficiency of thyroid hormone biosynthesis . Some inUnfortunately, it is not usually possible to distinguish individuals with permanent CH from those in whom CH will be transient at the time of diagnosis, although several studies have evaluated predictors of CH duration. The best indicators of TCH seem to be lower levothyroxine requirements throughout the course of treatment, lower TSH levels at diagnosis in comparison to those seen in permanent CH, and absence of TSH elevation during treatment , 23, 24.Based on the evidence to date, the suggested indications for treatment cessation include the absence of a definitive diagnosis of permanent CH, a daily dose of thyroxine ≤ 25 μg (consider if <3 μg/kg/day) and stable or decreasing dose requirements, without TSH rise on treatment  for iodination . Causes +/K+ ATPase. Specific transporters, for example, pendrin (SLC26A4) and anoctamin-1 then mediate iodide efflux into the follicular lumen, where it is oxidized in the presence of hydrogen peroxide (H2O2) and incorporated into tyrosyl residues on the surface of TG to form MIT and DIT. MIT and DIT couple to form thyroid hormones (T4 and triiodothyronine (T3) which are endocytosed back into the thyroid follicular cell and then cleaved and secreted into the circulation.During normal thyroid hormone biosynthesis, iodide is taken up from the circulation across the basolateral membrane of the thyrocyte by the sodium-iodide symporter  which coordinates the electrogenic symport of two sodium ions for one iodide ion, down an electrochemical gradient generated by the Na2O2-dependent oxidation of I− into I+, the iodination of tyrosyl residues, and the coupling of MIT and DIT. DUOX2 (an NADPH-oxidase enzyme) and its accessory protein, DUOXA2, are the predominant source of H2O2. Monocarboxylate transporter 8 (MCT8) and other transporters mediate TH efflux from the thyroid gland and iodotyrosine deiodinase (IYD) recycles unused iodide moieties  is more frequent than compatible with the prevalence of GIS CH. The true association of DUOX mutations with CH may be underestimated due to false-negative newborn screening results in mutation carriers if conventional (higher) TSH screening cut points are used or if the TSH rise is delayed . HoweverDUOX2, and not DUOX1, loss of function is associated with hypothyroidism; thus, the role of DUOX1 in thyroid biology remains unclear  are water-soluble and readily cleared from the body, fetal exposure to high iodide loads is transient. High osmolar, lipid-soluble ICM exhibit delayed excretion and confer a greater risk of hypothyroidism. A systematic review of neonatal thyroid function following the use of ICM before or during pregnancy found a tendency towards an increased risk of thyroid dysfunction with higher doses of contrast, especially when injected into the fetal compartment during amniofetography . HoweverNeonatal administration of ICM may also cause hypothyroidism, especially in premature neonates; in a systematic review, 8.2% of term infants and 18.3% of premature infants developed hypothyroidism after ICM exposure , 88. Indin utero exposure may cause neonatal TCH. Amiodarone is used for both maternal and fetal dysrhythmias, and over 20% of neonates exposed to amiodarone in utero exhibit hypothyroidism which may be biochemically profound and is sometimes associated with goitre; hypothyroidism may develop after only a few weeks of fetal exposure  is used most commonly as it carries the lowest risk of teratogenicity; however, later in pregnancy, methimazole (MMI), or its prodrug carbimazole, is sometimes used. Thionamide medication can cross the placenta, and since the kinetics of placental transfer are similar for PTU and MMI, they each carry a similar risk of causing fetal hypothyroidism or neonatal TCH , 93. SomGenerally, clinically evident fetal hypothyroidism is rare at low doses of PTU (≤50 mg daily) and a recent study showed no significant differences between maternal and cord FT4 levels at this dose . Both mein utero leading to permanent CH  which may be stimulating, blocking, or bio-inactive in nature. Different anti-TSHR antibody subtypes can coexist and can cross the placenta to the fetus from mid-gestation onwards , 97. A hanent CH .The clinician should become suspicious of the diagnosis when the pattern of levothyroxine dose requirements and thyroid biochemistry becomes inconsistent with athyreosis. A careful history and measurement of maternal thyroid function and antibody status may assist with early identification of this cause of TCH such that unnecessary prolonged treatment can be avoided and appropriate monitoring is undertaken in future pregnancies due to risk of recurrence .Artefactual TCH may occur due to transplacental passage of maternal antibodies which interfere with the TSH assay, causing spuriously high TSH results. Although FT4 and FT3 are usually normal in this context, significantly high TSH levels may result in misdiagnosis of CH and inappropriate levothyroxine treatment. This phenomenon may occur both with anti-animal or heterophile (poly-specific) antibodies targeting assay reagents and with anti-TSH antibodies resulting in the formation of macro-TSH complexes . TSH meaTransplacental passage of maternal-interfering immunoglobulins usually causes elevated TSH with normal FT4 and FT3 in a clinically euthyroid neonate, and a similar pattern of thyroid biochemistry is identified in the mother , 105. ItInfantile haemangiomas (IH) are the most common tumours of infancy, occurring in up to 10% of infants, and they follow a characteristic pattern of proliferative growth during the first year of life, followed by gradual involution. Diffuse infantile hepatic haemangiomas are frequently associated with consumptive hypothyroidism, due to high levels of type 3 iodothyronine deiodinase (D3) expression. D3 is a selenoenzyme, normally present in brain, placenta, and fetal liver and catalyses the conversion of T4 to reverse T3 (rT3) and the conversion of T3 to 3,3’-diiodothyronine, both of which are biologically inactive. In consumptive hypothyroidism, elevated TSH is associated with normal free T3 (fT3), normal or low-free T4 (fT4), and elevated serum rT3 levels as a result of increased T4 and T3 degradation by D3 , 107. ExTCH is increasing in incidence and, despite recognized genetic and environmental causes, there are no large-scale cohort studies investigating its basis and the reasons for this increase. Smaller studies have suggested the involvement of hitherto uncharacterized factors . ConsequThe most convincing potential contributors identified so far include the effects of increased ascertainment due to decreased screening cut points, the likely re-emergence of iodine deficiency in certain regions, and altered genetic background or ethnicity of the screened population, which may have changed over time. In some settings, increased survival of preterm infants may also contribute.All of these factors may interact; for example, DUOX-associated TCH may only be detected if screening cut points are low, iodine deficiency may unmask hypothyroidism in the setting of a mild dyshormonogenesis, and the preterm thyroid may be more vulnerable to genetic or environmental insult. However, there is a clear need for comprehensive studies evaluating genetic, environmental, and demographic factors in large TCH cohorts, in order to clarify the relative roles of these factors alone and in combination.Additionally, the long-term outcomes of children with TCH require evaluation, especially in regard to whether reintroduction of levothyroxine at time points of increased metabolic demand and growth may be required in children with underlying genetically mediated dyshormonogenesis. This may be most relevant during pregnancy where fetal development and pregnancy outcomes are dependent on euthyroid status.Finally, it remains disputed whether TCH always requires treatment, and it is sometimes not categorized as ‘true CH’ in epidemiological studies . Since Tin situ and may have lower than predicted thyroid hormone requirements. However, TCH cannot be predicted on the basis of initial neonatal screening biochemistry or thyroid scintigraphy. Moreover, although the association of TCH with DUOX2 and DUOXA2 mutations is well-recognized, CH duration cannot be predicted on the basis of genotype alone. Clinicians should assess whether a child with CH is likely to have permanent or transient CH by 3 years of age or earlier and trial individuals off levothyroxine therapy as indicated. When TCH is confirmed to be genetically mediated, the individual should be offered long-term monitoring for recurrent thyroid dysfunction, particularly during pregnancy.TCH is increasing in incidence, and although there are several well-defined genetic and environmental determinants, further studies are required to evaluate their relative contributions. Children with TCH will usually have a normally located thyroid gland The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.N S is funded by the Wellcome Trust (Grant number: 219496/Z/19/Z) and supported by the NIHR Cambridge Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.This research was funded in part, by the Wellcome Trust (Grant numbers 219496/Z/19/Z). For the purpose of open access, the authors have applied a CC-BY public copyright licence to any Author Accepted Manuscript version arising from this submission in accordance with the grant’s open-access conditions.


Ovarian cancer is one of the deadliest cancers in women and is unfortunately detected only in its later stages, by which time it will have metastasized to different organs. One of the major determinants of the rapid proliferation and spread of cancer is the presence of cancer stem cells (CSCs) within tumors. In this review, we discuss the emerging markers of ovarian cancer with respect to CSCs and circulating tumor cells. Cancer stem cell population is regulated by the Wnt signaling pathway, which is described in detail. This is relevant to comprehend the significance of Wnt in regulating the stem cell-like properties of cancer. Cancer stem cells are also responsible for enhancing chemoresistant properties and metastatic potential. Therefore, for a successful treatment regime, it is important to factor in drugs that are specific to CSCs. By targeting the promoters of CSCs, such as the Wnt signaling pathway, it is possible to suppress these cells specifically. Herein, we describe an inhibitor of Wnt, secreted frizzled related protein 4, which could be used to successfully destroy the cancer stem cells of ovarian cancer.Ovarian cancer is one of the most prevalent gynecological cancers, having a relatively high fatality rate with a low five-year chance of survival when detected in late stages. The early detection, treatment and prevention of metastasis is pertinent and a pressing research priority as many patients are diagnosed only in stage three of ovarian cancer. Despite surgical interventions, targeted immunotherapy and adjuvant chemotherapy, relapses are significantly higher than other cancers, suggesting the dire need to identify the root cause of metastasis and relapse and present more precise therapeutic options. In this review, we first describe types of ovarian cancers, the existing markers and treatment modalities. As ovarian cancer is driven and sustained by an elusive and highly chemoresistant population of cancer stem cells (CSCs), their role and the associated signature markers are exhaustively discussed. Non-invasive diagnostic markers, which can be identified early in the disease using circulating tumor cells (CTCs), are also described. The mechanism of the self-renewal, chemoresistance and metastasis of ovarian CSCs is regulated by the Wnt signaling pathway. Thus, its role in ovarian cancer in promoting stemness and metastasis is delineated. Based on our findings, we propose a novel strategy of Wnt inhibition using a well-known Wnt antagonist, secreted frizzled related protein 4 (sFRP4), wherein short micropeptides derived from the whole protein can be used as powerful inhibitors. The latest approaches to early diagnosis and novel treatment strategies emphasized in this review will help design precision medicine approaches for an effective capture and destruction of highly aggressive ovarian cancer.  Ovarian cancer is the most lethal gynecological malignancy, with a 5-year survival rate of only 17% in the advanced stages . Most paOvarian cancer, despite its high lethality and low survival rate, does not have precise identification markers. Cancer stem cells are the key factor determining the drug response of the tumors and are usually identified by specific bonafide markers. In this review, we discuss the types of ovarian cancer and the existing diagnostic markers, chemo resistance conferred by CSCs and a unique panel for the identification of ovarian CSCs and circulating tumor cells. The role of Wnt in ovarian cancers, promoting stemness and metastasis, is a crucial factor in designing drug targets. An emerging antagonist of Wnt, secreted frizzled related protein 4 (sFRP4), has been recently found to be a promising agent to suppress CSCs. Could the functional peptides of sFRP4, in targeting ovarian cancers and their aggressive CSC population, evolve into a promising peptide drug panel for treating highly malignant and drug-resistant ovarian cancer? In this review, an overview of the existing challenges and evolving promising approaches and trends for ovarian cancer treatment is addressed.Most tumors from the ovary develop from one of the three components: the surface epithelium, germ cells and sex cord stroma. Epithelial ovarian carcinomas (EOC) account for the maximum number of cases and these are histologically subdivided broadly into five categories ,6,7,8,9 The definitive etiology and risk factors involved in the progression of ovarian cancer are not well studied but a few theories have been postulated, the majority having genetic links. Nulliparous women and those with longer reproductive years, i.e., early menarche and/or late menopause, are known to be at a higher risk for ovarian malignancy. On the contrary, multiparity and the use of oral contraceptive pills (OCPs), especially in pre- menopausal women and tubal ligation, prove to be protective factors. This is backed up by the incessant ovulation theory, which was hypothesized through a case control study conducted by Casagrande et al., where patients with factors accounting for “protected time”, i.e., longer periods of anovulation, through pregnancies and the use of OCPs had a significant reduction in the risk for developing ovarian cancer . Among the epithelial ovarian cancers, the endometrioid and the clear cell type are known to be derived from ovarian endometriosis. A widely accepted theory is that of retrograde menstruation, which enhances iron-induced oxidative stress and in turn DNA damage. Some studies have even shown that gene mutations associated with endometriosis-associated ovarian cancers are normally found in the uterine and ovarian endometriotic epithelium . High-grAmong the various cancer-detecting strategies, tumor markers have been used for decades as a starting point in the roadmap for cancer diagnosis. They are specific proteins and biomarkers released in blood in response to carcinogenesis and other inflammatory states. First identified by Bast et al. in 1981, CA-125 is the oldest and most widely used biomarker for epithelial ovarian cancer ,17. AlthHE4 (WFDC2) is a glycoprotein belonging to the family of whey acidic proteins (WAP) and contains two WAP domains. It was initially isolated in human epididymis by Kirchhoff et al., but its presence outside the male reproductive system was noted eventually in further studies. Although it was weakly expressed in lung and colorectal adenocarcinoma, it was found to have significant expression in epithelial ovarian tumors, especially in the endometrioid subtype .Statistical analysis showed a sensitivity of 72.9% for the HE4 marker alone when a comparative study between other markers such as CA-125, CA72-4, osteopontin, soluble mesothelin-related peptide and human epidermal growth factor 2 was performed by Moore et al., while Yanaranop and his team reported a specificity of 86% ,20,21. SMoore and his team in 2009 proposed the Risk of Malignancy Algorithm (ROMA), which took into account HE4, CA-125 and the age of a patient presenting with a pelvic mass  and clasProstatin (PSN), a trypsin-like protease normally found in prostatic and seminal fluids, was also studied as a potential biomarker when Costa et al. observed a significant overexpression in prostatin mRNA in epithelial ovarian cancers . The comTranserythrin (TTR) has been found to be useful for the detection of stages I and II of epithelial ovarian cancer. Alpha fetoprotein (AFP) and beta HCG are used to detect the stages and treatment response of ovarian germ cell tumors. Inhibin A and B were found to be overexpressed in mucinous epithelial cancer and granulosa cell tumors ,25.Treating ovarian cancer requires a personalized and multi-disciplinary approach, taking into account the stage at which the patient is diagnosed  and their co-morbidities. A prospective study demonstrated a statistically significant difference in the benefit of chemotherapy and overall prognosis in patients whose cancer was optimally staged . This reDebulking cytoreductive surgery, which consists of total abdominal hysterectomy (TAH) + bilateral salpingo-oophorectomy (BSO) followed by three to six cycles of chemotherapy, is the initial step in the management of OC, recommended by the Gynecologic Cancer Inter-Group (GCIG) . Nodal mUnfortunately, 75% of patients are diagnosed for the first time in advanced stages (FIGO stages III and IV) of OC or patients have returned with recurrent disease that has metastasized extensively. This has prompted physicians to adopt a more aggressive approach, i.e., pre-operative imaging followed by maximal cytoreduction. A meta-analysis by Bristow et al.  elucidatThe role of PARP inhibitors as a single therapeutic agent is most beneficial in HGSOC patients with BRCA mutations. PARP 1 plays an important role in DNA single stranded breakage (SSB) and double stranded breakage (DSB) repair through base excision repair mechanisms. BRCA genes, among other proteins in normal cells, are involved in the homologous recombination repair (HRR) of DSB. The inhibition of PARP in the presence of BRCA 1 and 2 mutations results in defective HRR and DSB repair leading to genomic instability and cell death. This is referred to as ‘synthetic lethality’, wherein the inactivation of two pathways simultaneously evokes a strong and selective apoptotic response in cancer cells ,34. The The vascular endothelial growth factor (VEGF) pathway is the most common target for angiogenesis inhibition in many cancers including OC. Bevacizumab (avastin) is a monoclonal antibody that targets the VEGF pathway and is approved for use in advanced OC patients . Over tiEpithelial OC is considered an inflamed tumor, one that has a high percentage of CD8+ T cells and high PD 1 expression . This plAlthough ovarian cancer is known to be radiosensitive, the role of whole abdominal radiotherapy (WAR) is limited in standard care after the introduction of platinum-based chemotherapy . This isComplete remission is achieved with platinum chemotherapy and debulking surgery in a majority patients, but most of them experience a relapse . DespiteCancer stem cells (CSCs) are a small subset of the stem cell population with properties such as self-renewal, differentiation and resistance to apoptosis, similar to tissue stem cells . The disThe presence of tumor-initiating cells in malignancies was initially discovered by Lapidot et al. in 1994 when they found the increased expression of certain cell surface markers  in relation to acute myeloid leukemia in an immunocompromised state . Over tiCancer stem cells are identified by their unique profile of cell surface and intracellular markers. The panel of ovarian CSC markers that are reported include CD24, CD44, CD117, CD133, ALDH1, ABC transporter proteins, EpCAM, Nestin, Oct4, Nanog, Sox2, SSEA4 and SCF. Of these, CD24, CD44 and CD133 are a few of the prominent markers. CD24+ cells in ovarian cancer exhibit anoikis resistance, higher tumor growth, colony formation, EMT phenotype and possess stem-like properties such as self-renewal. CD133 expression is associated with poor prognosis and is also implicated in increased platinum resistance and metastasis. Nanog expression also modulates platinum resistance and EMT in ovarian cancer. A comprehensive list of ovarian-specific CSC markers and their role is summarized in The proliferative potential of CSCs is regulated by the tumor microenvironment and multiple pathways. Heterogeneity in the tumor microenvironment is a classic feature of ovarian cancer and the distinct mutational profile of epithelial ovarian cancer is what makes it unique and challenging among other cancers. The clonal evolution theory was proposed in 1976 , where lMultiple regulatory pathways such as Wnt, STAT3, Hedgehog, BMI1, Notch and NF-κB are abnormally activated and thus aid in the self-renewal of CSCs in ovarian cancer ,92. The The presence of circulating tumor cells (CTCs) in ovarian cancer is gaining clinical relevance . CirculaWnts are cysteine-rich glycoproteins secreted in the extracellular matrix. The Wnt pathway is a highly conserved evolutionary system, existing across species from invertebrates to mammals, and is responsible for various cellular processes such as proliferation, differentiation, apoptosis, tissue homeostasis and stem cell renewal . NineteeThe canonical Wnt pathway is dependent on the dual function protein, beta catenin. In the absence of a Wnt ligand, the cytoplasmic beta catenin is phosphorylated by the actions of adenomatous polyposis coli (APC), Casein kinase 1 alpha (CK1α) and Glycogen synthase kinase 3β (GSK3β) present in Axin (a scaffold protein that constitutes the destruction complex) ,107. ThiThe initial discovery of Wnt canonical signaling was associated with its role in tumorigenesis when the transcriptional activation of molecule Int 1 was found to induce mammary hyperplasia in mice through pro-viral insertion into the Wnt 1 locus ,111. FurThe regulation of ovarian tumorigenesis by Wnt normally occurs extracellularly through its antagonists ,120. In sFRP4 is a glycoprotein modulator and the largest member of the sFRP family. It contains two domains: a cysteine-rich domain (CRD) that is homologous to the Wnt binding site of frizzled proteins and a netrin-like domain (NLD). It has apoptotic and anti-angiogenic properties, thus acting as a tumor suppressor . sFRP4 wIn our recently published report , it was To summarize, in this review we describe the existing prognostic and diagnostic markers in ovarian cancers and their level of specificity. The treatment strategies currently adopted by physicians globally shed light on the need for a more effective and precise panel of drugs, since relapses still remain alarmingly high. The significance of cancer stem cells in promoting chemoresistance and metastasis and the specific markers of CSCs and CTCs are described. The role of Wnt in regulating CSCs is further elaborated as the Wnt signaling pathway is one of the major determinants of the survival of CSCs. Novel targeted therapy addressing upregulated Wnt signaling would be the way forward to inhibit ovarian cancer stem cells. A natural antagonist of Wnt, the secreted frizzled related protein 4 has been well reported to increase chemotherapeutic response and decrease the CSC population. We describe how the short active peptide derivatives of sFRP4 could be the latest breakthrough in peptide-based drug compounds for the treatment of highly malignant and aggressive ovarian cancer.


Adult neurogenesis represents a striking example of neural plasticity throughout life where adult-born neurons are continuously generated from neural stem cells (NSCs). The hippocampus is a neurogenic niche where postnatal neurogenesis occurs, where the role of neurogenesis in this brain region is to maintain healthy hippocampal function, such as learning and memory across the lifespan  could control neuronal differentiation without affecting the proliferation of hippocampal NSCs. They also showed that while HDAC1 and HDAC2 was expressed in all stages of adult hippocampal neurogenesis, stem/progenitor cells exhibited a higher tendency for HDAC1 expression, whereas immature granule neurons had more HDAC2 expression. However, conditional HDAC1 knockout-mediated influence on the neuronal differentiation was not reproduced in an in vivo system, suggesting that more complex mechanisms are at play regarding the role of HDAC1 in adult neurogenesis.During the process neuronal differentiation and maturation, NSCs/neural progenitors require coordinated changes in the pattern of gene expression. While epigenetic regulatory mechanisms have emerged as a critical regulator to fine-tune and coordinate gene expression during adult hippocampal neurogenesis  recordings showed no differences in the formation of spontaneous recurrent seizures in the two groups, showing that IL-17A does not play a major role in epileptogenesis. Using histological methods, the authors found that IL-17A KO mice showed reduced neuronal death and fewer ectopic granular cells in the hippocampus without a difference in cell proliferation or number of immature neurons in the subgranular zone of the dentate gyrus. Together, their results indicate a role of IL17-A in mediating TLE-associated anxiety behavior possibly by reducing ectopic neurogenesis and neuronal death in the hippocampus.Dysregulation of adult hippocampal neurogenesis contributes to a variety of brain diseases  in a model of oxidative stress using both sequencing paradigms. They found that both methods resulted in little overlap in common DEGs, and the DEGs identified by sc-RNAseq were from genes that had relatively higher transcript counts and had smaller fold changes compared to the DEGs identified by bulk seq. The authors then took advantage of their two data sets of the same population of cells to develop a workflow that can determine what input of RNA is necessary to preserve unbiased detection of DEGs. Using this approach, the authors were able to determine an RNA-input threshold that generated transcriptional profiling results of NSCs comparable with bulk sequencing. This approach is a great resource for researchers studying rare cell types in vivo.Advances in sequencing technologies has allowed to transcriptionally distinguish populations of cells involved in adult neurogenesis . L-serine is an indispensable neurotrophic factor and a precursor for neurotransmitters. While the significant role of L-serine in preventing neuroexcitotoxicity, neuroinflammation, and cerebral blood flow associated with brain injuries  are comprehensively discussed, this review also highlights L-serine's role in adult neurogenesis and oligodendrogenesis as potential neuroprotective mechanisms. For instance, L-serine promotes the survival, proliferation and differentiation of NSCs. In addition, L-serine also stimulates the proliferation and survival of oligodendrocytes derived from NSCs following brain injury, which plays an important role in myelin recovery. Collectively, L-serine serves as a potential regenerative agent that stimulates adult neurogenesis as well as oligodendrogenesis, and may represent a therapeutic option to facilitate brain repair.This Research Topic also discussed the role of L-serine as a potential neuroprotective agent for neurological disease and brain injury  BB/W008068/1 to DB, National Research Foundation of Korea (NRF) (2019R1A2C1003958 and 2021R1A4A5028966) to K-OC, and NIH (R01CA242158 and R01AG058560) to M-HJ.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The rapid evolution of genomic technologies over the last years has led to the development of different methods for the detection, measurement and analysis of cell-free DNA fragments (cfDNA) which are shed into the bloodstream by apoptotic cells and circulate at a low concentration in plasma. In cancer patients, the proportion of tumor-derived cfDNA is defined as circulating tumor DNA. This analysis, commonly known as liquid biopsy, allows to access tumor DNA through a simple blood sampling and therefore without the need of an invasive tissue biopsy. For this reason, this tool may have several clinical applications in terms of diagnosis, prognosis, and monitoring of minimal residual disease. However, there are still several critical issues that need to be resolved. In this review, we will discuss some of the controversies around this method and its potential clinical applications. Historically, the blood of patients with leukemic hematological malignancies has represented a source of circulating tumor cells (CTC), thus constituting an opportunity for simple, noninvasive analysis of their genetic profile.4 but only in 1977, it was recognized that patients with malignancy have higher levels of cfDNA compared with healthy individuals.5 A potential role of cfDNA in profiling a tumor genetically was first demonstrated in 1994 following the identification of tumor-associated RAS gene mutations in the plasma of patients with myelodysplastic syndrome and acute myeloid leukemia.7 Several years after, Lawrie et al7 showed for the first time that tumor-associated microRNAs are also detectable in the peripheral blood of lymphoma patients. These discoveries led to an increasing number of studies that, using more sensitive methods, investigated the utility of cfDNA for the characterization, monitoring and therapeutic targeting of both hematologic and solid malignancies.8Along with cells and proteins, blood also contains fragments of nucleic acids that circulate free in its plasma fraction. Circulating cell-free DNA (cfDNA) was discovered in 1948 in the blood of healthy individuals,9 Moreover, ctDNA analysis identifies mutations, amplifications, deletions and translocations, and allows the detection of genetic alterations associated with response to treatment, also known as predictive biomarkers.10 In this field, the milestone was represented by the Food and Drug Administration (FDA) approval in 2016 of the first blood ctDNA test based on specific epidermal growth factor receptor (EGFR) mutations, able to guide the use of EGFR-tyrosine kinase inhibitors in patients with non–small-cell lung cancer.11 More recently, other assays were approved by FDA namely companion diagnostic tests, including assays for the detection of PIK3CA mutations in breast cancer.12Accessing cancer DNA through a venipunction grants several opportunities beside its obvious convenience for both patients and physicians. Compared with the cellular fraction of blood, cfDNA is a more abundant source of tumor genetic material in cancers lacking CTC as several solid tumors and lymphomas. Compared with circulating protein biomarkers, cfDNA is more specific because it harbors the tumor fingerprint made up by its somatic mutations. Compared with single biopsies in primary cancer sites, cfDNA can be longitudinally assessed and can provide a more complete description of the cancer mutational profile; this may be of particular importance in the case of spatially and genetically heterogeneous malignant tumors. The rapid evolution of genomic technologies over the past 2 decades has led to the development of methods for the measurement and analysis of circulating tumor DNA (ctDNA) that supported a significant progress toward potential clinical applications. CtDNA analysis provides quantitative information about the tumor burden and is useful for detection of minimal residual disease (MRD) and occult metastases.While ctDNA technologies have entered in the management of a limited number of solid tumors, their analytical and clinical validation in lymphoma still remains a critical open question. In this controversy article, we aim to provide an overview of the currently debated aspects around: (i) the physiology and pathophysiology of cfDNA in lymphoma; (ii) the impact of preanalytics on ctDNA assay results in lymphomas; (iii) the technical validity, and the real-time feasibility of state-of-the-art ctDNA assays; and (iv) the clinical utility of state-of-the-art ctDNA assays to guide lymphoma diagnosis, treatment tailoring, and residual disease identification.13 The amount of cfDNA in plasma corresponds to hundreds genomic equivalents per ml of plasma and is highly fragmented. The size of cfDNA varies from ~40 to 200 base pairs (bp), with a peak at approximately 166 bp. On its release, cfDNA is fragmented and nucleosomes, transcription factors, and other DNA binding proteins prevent random cleavage, resulting in specific patterns of fragmentation. These fragmentation profiles reflect chromatin proteome occupancy maps and epigenetic fingerprints. Epigenomic footprinting through both nucleosome occupancy inferred from cfDNA fragmentation patterns and methylation profiling point to the hematopoietic lineages as a major source of cfDNA in healthy subjects.18cfDNA refers to DNA that is freely circulating in plasma. In patients with cancer, the proportion of tumor-derived cfDNA is termed ctDNA. Various forms of cell death, including apoptosis, are the predominant source of ctDNA.19The distinction of ctDNA from background cfDNA released by hematopoietic cells may be confounded by biological signals arising from clonal hematopoiesis. Clonal hematopoiesis is a common aging-related phenomenon where somatic mutations in hematopoietic stem cells are clonally propagated to their progeny. High-sensitivity, deep next-generation sequencing (NGS) approaches used in ctDNA mutation recovery can identify biological signals from clonal hematopoiesis in up to 90% of cancer patients and must be distinguished from tumor associated mutations. Consistently, a large fraction of cfDNA mutations recovered in cancer patients have features consistent with clonal hematopoiesis, which in turns can result in inaccurate ctDNA quantification and mutational signature recovery. Joint analysis of cfDNA and matched leukocytes distinguishes background mutations due to clonal hematopoiesis and allows accurate variant interpretation and recovery of lymphoma-associated mutations.20The concentration of cfDNA in blood varies significantly. It ranges between 0 and 100 (median ~10) ng/mL in healthy subjects, and is usually elevated, though with great variability, in patients with lymphoma, ranging from 5 and 100 (median ~25) ng/mL. The fraction of lymphoma ctDNA constitutes ~0.1%–90% (median ~5%) of cfDNA. Therefore, in most lymphoma patients, a minor part of total cfDNA consists of ctDNA, though there is a great variability in the ctDNA/cfDNA ratio depending on tumor bulk and histology. Indeed, the ctDNA/cfDNA ratio is higher in patients with larger tumor volumes and/or aggressive histologies compared with patients with low tumor volume and/or indolent histologies. Given the typical low ctDNA/cfDNA ratio, highly sensitivity wet-lab approaches and bioinformatics pipelines are needed for the accurate quantification and mutation profiling of ctDNA.21 Consistently, levels of ctDNA also vary according to different histological subtypes of lymphoma, with higher levels in aggressive lymphomas such as in diffuse large B-cell lymphoma (DLBCL), classical Hodgkin lymphoma (cHL), and mantle cell lymphoma (MCL) in comparison to follicular and other indolent lymphomas which have circulating DNA levels similar to healthy controls.24 Compared with solid tumors, several lymphoma types release higher amounts of ctDNA.27 Stage of disease and tumor burden have also an impact on cfDNA levels, which are higher in advanced stage lymphomas than in limited stage disease, and in overt progressive disease than in a disease in clinical response after the treatment.29 Collectively, these data indicate a relationship between tumor burden and extension and ctDNA release. Aggressive tumors with rapid turnover of proliferation and apoptosis usually have the highest levels of ctDNA , the concentration of ctDNA varies considerably and it may also depend on the ratio of tumor malignant cells to tumor microenvironment cells.31 This effect has direct implications on the collection timing following lymphoma treatment when ctDNA is used as a surrogate biomarker of tumor reduction.A systematic analysis of the best timepoint for measuring ctDNA after tumor treatment is lacking. Release of ctDNA into the circulation depends on the location, size, and vascularity of the tumor, leading to a difference in ctDNA levels among patients. cfDNA is quickly cleared from plasma by organs, such as the liver, spleen, kidney. Therefore, cfDNA has a short and variable half-life, which ranges from 16 minutes to 2.5 hours.32 In one study that evaluated the efficacy of panobinostat in DLBCL, a significant increase of ctDNA on day 15 as compared with baseline was associated with lack of response to treatment.33 A phase I/II trial investigating anti-CD19 CAR-T cells in DLBCL showed that ctDNA levels assessed at 28 days after the product infusion were predictive of outcome.34 Therefore, the identification of the best timepoint to evaluate ctDNA is still a controversy, and likely depends on the type of underlying disease, type of treatment and its scheduling . There are several different cfDNA purification methods, with different performance that may affect cell-free DNA yield and purity.36 Methodological studies should accurately measure differences in yield and quality of cfDNA and ctDNA by comparative testing of multiple methods and protocol parameters with the same plasma input.Unsolved issues in the preanalytical handling of cfDNA have been discussed by a position paper of the American Society of Clinical Oncology and College of American Pathologists.−5) and high specificity.37 A drawback is that they require a priori knowledge that the mutation specifically investigated by the assay is specific for the tumor.Polymerase chain reaction (PCR)-based methods can study a single mutation at a known locus. Advantages of PCR-based methods are high sensitivity , primary central nervous system lymphoma (PCNSL), and vitreoretinal DLBCL. In WM, Drandi et al38 showed that there was a good correlation of the detection of MYD88 L265P mutation on plasma ctDNA and bone marrow findings.An example of the application of droplet digital PCR (ddPCR) is the detection of the MYD88 L265P mutation has a sensitivity rate of 60% in plasma samples39 and could potentially be used on cerebrospinal fluid (CSF) samples.In PCNSL, a ddPCR assay probing the hotspot MYD88 L265P mutation in vitreous fluid (another kind of liquid biopsy) with high sensitivity.40Finally, in vitreoretinal DLBCL, ddPCR can detect the 42Another example of the possible application of ddPCR is in cHL, where the detection of XPO1 hot spot by ddPCR may be used as biomarker for diagnosis and MRD.−5.10 All these methods present technical challenges and their potential applications currently represent a field of intense investigation in lymphoma research.NGS-based assays are able to detect immunoglobulin and T-cell receptor gene rearrangements, multiple classes of gene mutations, rearrangements, and copy number alterations. They are off-the-shelf approaches that do not require a prior knowledge of the mutation profile and clonality of the tumor and allow the identification of novel mutations occurring during clonal evolution of a tumor. Sensitivity of NGS assays incorporating chemistries or bioinformatics that suppress the error rate is comparable to that of PCR-based approaches, with a detection limit of 1043Among these methods, immunoglobulin high-throughput sequencing (Ig-HTS) involves identification and tracking of the unique immunoglobulin sequence from the malignant B cells by using universal primers that target immunoglobulin genes. These methods have been studied in a variety of lymphoma subtypes, including DLBCL and MCL.45Ig-HTS can track the clonal heterogeneity and clonal evolution in lymphomas comparing immunoglobulin somatic mutations at diagnosis and during the follow-up as demonstrated in FL and DLBCL, although when tumor IgH sequences are not available, IgH-HTS may be less performant.46 This method utilizes a disease-specific selector (set of exonic and intronic targets chosen to cover regions of known recurrent mutations) to selectively sequence genomic regions of interest in a patient sample. CAPP-seq can in theory simultaneously test all important classes of mutations, including single nucleotide variants (SNVs), insertions/deletions (indels), copy number alterations and rearrangements.47 Given its high sensitivity and ability to reliably identify patient-specific target sequences, this NGS assay has been used in highly promising studies in lymphomas.48The CAncer Personalised Profiling by deep Sequencing (CAPP-seq) is a sensitive tool used to detect disease-specific mutations in ctDNA.49  which can convincingly detect somatic copy number aberrations. This method is less sensitive but does not require prior information on the tumor genome and allows identification of new mutations which may develop during treatment9 Figure .As mentioned before, 2 strategies have been developed to study ctDNA: the first is based on querying tumor specific mutations and uses assays characterized by high sensitivity and specificity but analyzing a limited number of known mutations (PCR-based approach), while an untargeted approach performs a more extensive study of the genome but with low sensitivity and requiring high amounts of ctDNA (NGS-based approach).50 recently described phased variant enrichment and detection sequencing (PhasED-seq) method, which is able to identify multiple somatic mutations in individual DNA fragments improving the sensitivity of ctDNA detection. Alternative bioinformatic approaches utilizing unique molecular identifiers in combination with bioinformatic error reduction using individual base error likelihoods as well as error likelihoods in base triplets have been proposed.51The limited sensitivity of the NGS methods used for the detection of ctDNA is a critical aspect for the use of this tool for the monitoring of MRD. To solve this problem, several bioinformatic pipelines have been developed to increase the sensitivity of mutation detection and decrease the sequencing error rate, in particular for monitoring residual disease. Kurtz et alIn the context of MRD study where tumor fraction is low, deep sequencing methods with limited genomic targets have been applied. However, many patients with radiologically evident disease after treatment do not show detectable ctDNA. Deep-targeted NGS approaches are hindered by a fundamental barrier to detection sensitivity, namely the limited input material . This may be particularly relevant in the settings of early cancer or MRD detection. In a typical plasma sample, cfDNA fragments are often in the range of only hundreds to several thousand genomic equivalents per milliliter. The limited number of genomic equivalents effectively places a ceiling on the depth of sequencing beyond which available distinct fragments are exhausted. The limited cfDNA input cannot be overcome through improvements of sample processing. Therefore, the only way of increasing cfDNA input would be maximizing blood collection from patients.−4). In DLBCL, the total number of somatic SNVs detected per case range from 1.000 to 50.000, with an average of 12.000 somatic mutations.52 Consistently, Zviran et al36 demonstrated in clinical cohorts of lung cancer, CRC, and melanoma that the use of WGS of cfDNA with a modest sequencing depth (ca. 30×) allowed ultrasensitive detection with a sensitivity of 10−5 demonstrating that breadth can overcome the depth of sequencing.The sampling limitation can be effectively overcome by increasing the chance of detecting a ctDNA fragment through increased breadth of sequencing. Indeed, if the number of interrogated sites is wide as the genome, the chance of detecting a mutant ctDNA becomes dependent on the total number of somatic mutations within a typical tumor genome rather that the depth of sequencing  was higher in cfDNA from peripheral blood compared with genomic DNA from bone marrow or random skin biopsies.55 Futhermore, Shimada et al studied provided additional insights on the molecular pathogenesis of IVLBCL, identifying among others PD-L1, PDL2 and other genes in cfDNA.57 These data demonstrate that ctDNA analysis is complementary to clinical and pathological results and supports its potential usefulness for the diagnosis of lymphoma residing in poorly inaccessible anatomical sites.Another difficult diagnostic setting is represented by intravascular large B-cell lymphoma (IVLBCL), a rare lymphoma entity whose conventional diagnosis is based on the presence of lymphoma cells infiltrating blood vessels. In a small series of patients with IVLBCL among 9 patients with paired (tissue and plasma) samples available, allele frequency of mutations detected by NGS , and total metabolic tumor volume.Genotyping of baseline ctDNA provides an average of genetic information within the tumor that may complement or replace information from tissue biopsies, which may be relevant if actionable mutations are used as predictive biomarkers for treatment tailoring.60 Kurtz et al29 have shown in a large study that ctDNA was detectable by CAPP-Seq in 98% of DLBCL patients at baseline and pretreatment ctDNA levels highly correlated with disease burden. Moreover, they found an optimal threshold to predict outcome; a 2-log drop in ctDNA after 1 cycle (defined as early molecular response [EMR]) and a 2.5-log drop after 2 cycles (defined as major molecular response [MMR]) predicted Event-free survival in 2 validation sets of patients in front-line therapy.29A challenging question in the management of patients with lymphoma is how to monitor residual disease with high enough sensitivity, both during treatment and in post-treatment surveillance. ctDNA measurement allows for dynamic monitoring of residual disease, which complements and enhances conventional imaging  in lymphoma restaging.61In this context, it has been proposed the continuous individualized risk index, a method based on the integration of 6 complementary risk predictors, including 3 established risk factors  and 3 ctDNA risk factors  to dynamically determine the outcome of patients with DLBCL.32 Another study in 121 patients showed in a cohort consisting of early and advanced stage classical HL patients that MRD measured by ctDNA reduction can differentiate good and bad outcomes to treatment as early as 1 week after initiation of treatment.51 ctDNA is being investigated in lymphoma and in particular in DLBCL and HL as a tool that could allow early and accurate identification of chemorefractory patients who are candidates for treatment intensification, or the identification of good-risk patients, for treatment de-escalation.In classical HL, a 2-log-fold reduction in ctDNA levels measured by CAPP-Seq after 2 cycles of doxorubicine, bleomycine, vinblastine, dacarbazine in patients with advanced stage disease was also strongly associated with PFS; conversely, a drop of less than 2-log in ctDNA after 2 treatment courses is associated with an eventual progression.34 In another study, a significant increase in ctDNA at day 15 strongly associates with lack of response in lymphoma patients treated with panobinostat.33On the other hand, the use of ctDNA for measuring remaining disease at an early interim timepoint may represent an early marker of treatment resistance in DLBCL patients treated with a chemo-free regimen. For example, ctDNA levels at day 28 after CAR-T cell infusion were shown to be prognostic in DLBCL.28Post-treatment surveillance has typically been performed by conventional scans , but several reports have highlighted the high sensitivity of liquid biopsies to detect disease recurrence before there is clinical evidence of relapse. An analysis of serial serum samples and concurrent CT scans during 5 years of follow-up in patients with DLBCL showed that resurgence of detectable ctDNA during follow-up strongly associates with subsequent clinical disease progression, with a median time of 3.5 months before CT scan positivity.Baseline ctDNA load can replace current staging criteria only if it is documented to outperform modern imaging techniques that are conventionally used for tumor load estimation. Likely, ctDNA load will complement, but not substitute, baseline imaging, as it may capture biological features that are masked to imaging. Tumor aggressiveness is characterized by uncontrolled proliferation that is associated with high levels of apoptosis. As a consequence, aggressive tumors with rapid turnover proliferation and apoptosis are more prone to release high levels of apoptosis-derived ctDNA into the blood. In this scenario, ctDNA may vary considerably within the same disease stage of a given lymphoma type and herald prognostic information in addition to tumor volume.MYD88 L265P and CXCR4 mutations in WM, EZH2 mutation in FL, TP53 mutation in small lymphocytic lymphoma) mutation profile of the tumor does not affect treatment decisions  and consensus on the most sensitive approach to be carried forward  are urgently needed to ensure accurate and reproducible detection across centers Table .Despite positive results, the current literature on MRD detection in cfDNA is based on retrospective cohorts. Prospective studies are now needed to establish the feasibility of real-time ctDNA evaluation for diagnosis and follow-up as well as the most appropriate timing of collection. It will need to be determined how ctDNA integrates with established imaging for complementary monitoring of treatment response, and how to measure the utility of ctDNA as an earlier surrogate endpoint and in risk-adapted clinical trials.ctDNA is a promising and emerging lymphoma biomarker and represents a potential tool for diagnosis, prognosis, and monitoring of disease. The main critical aspect remains sensitivity and the lack of standardization of preclinical techniques, the need for technological standardization and harmonization, a consensus on the threshold nominating high ctDNA levels in pretreatment samples, and MRD negativity during/after treatment. At present, there are no published data on prospective studies based on a liquid biopsy-driven approach in lymphoma patients but some of them are ongoing for clinically validating ctDNA in lymphomas  with the aim of studying ctDNA as a baseline biomarker to identify high-risk patients and also to define its role as a tool for disease response assessment. Further technological assessments and clinical studies will be required to validate this tool.This work was supported by the grant: “Molecular bases of disease dissemination in lymphoid malignancies to optimize curative therapeutic strategies”, (5 x 1000 No. 21198); Associazione Italiana per la Ricerca sul Cancro Foundation Milan, Italy. SB received Deutsche Forschungsgemeinschaft (DFG) grants EN 179/13-1 and BO 5316/2-1, the HL MRD consortium, and the Frauke-Weiskam und Christel Ruranski-Stiftung.SB is a shareholder founder and CEO of Liqomics, a liquid biopsy company. GG is in the advisory board of Abbvie, Astra-Zeneca, Beigene, Incyte, Janssen, Roch and speaker’s bureau Abbvie, Janssen. DR received honoraria from Janssen, AbbVie, and AstraZeneca, and research grants from Janssen, AbbVie, and AstraZeneca. All the other authors have no conflicts of interest to disclose.


Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by the PRRS virus that leads to reproductive disorders and severe dyspnoea in pigs, which has serious economic impacts. One of the reasons PRRSV cannot be effectively controlled is that it has developed countermeasures against the host immune response, allowing it to survive and replicate for long periods. Transcription Factors acts as a bridge in the interactions between the host and PRRSV. PRRSV can create an environment conducive to PRRSV replication through transcription factors acting on miRNAs, inflammatory factors, and immune cells. Conversely, some transcription factors also inhibit PRRSV proliferation in the host. In this review, we systematically described how PRRSV uses host transcription factors such as SP1, CEBPB, STATs, and AP-1 to escape the host immune system. Determining the role of transcription factors in immune evasion and understanding the pathogenesis of PRRSV will help to develop new treatments for PRRSV. Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRSV that leads to typical reproductive disorders and respiratory diseases in pigs , which are the first line of defense during the occurrence of infections. MiRNAs also play a crucial role in mediating host acquired immunity; however, their abnormal expression results in the manifestation of diseases  is a well-known member of the transcription factor family that is involved in a large number of important biological processes and can activate the transcription of many cellular genes  are a family of transcription factors that includes 9 members. It regulates many aspects of innate and adaptive immune responses, including driving antiviral responses, responding to pathogens, driving proinflammatory responses and regulating immune cell differentiation  protein  family transcription factors are closely related to inflammation observed in various viral and traumatic diseases. IL-17 is a proinflammatory cytokine that is closely related to the strong inflammation caused by PRRSV. Research has shown that PRRSV nsp11 is involved in IL-17 production and PRRSV replication. CEBPB exacerbates lung inflammation by promoting IL-17 production during induced PRRSV infection. In porcine IL-17 promoters, deletion of CEBPB and the CREB binding motif inactivated PRRSV-induced IL-17 production, while deletion of CEBPB and CREB significantly reduced PRRSV-induced IL-17 production, suggesting that IL-17 expression is dependent on CEBPB and CREB . Phosphorylation of IRF3 is a crucial step in the induction of IFNs  is a nuclear protein that is involved in precursor mRNA splicing. Studies have shown that PRRSV can significantly promote the expression of RBM39 in PRRSV-infected 3D4/21 cells . Numerous reports have demonstrated that CD4+CD25+Tregs can inhibit host antiviral immune responses. Induction of CD4+CD25+ Tregs during an early stage of infection has been suggested to be the mechanism for developing chronic or persistent viral infections  of nsp11 being responsible for STAT2 degradation, and also with the STAT2 NTD and core-coil domain  and Henan Province Scientific and Technological Key Project (222102110445 and 222102110477).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Systemic and pulmonary circulations constitute a complex organ that serves multiple important biological functions. Consequently, any pathological processing affecting the vasculature can have profound systemic ramifications. Endothelial and smooth muscle are the two principal cell types composing blood vessels. Critically, endothelial proliferation and migration are central to the formation and expansion of the vasculature both during embryonic development and in adult tissues. Endothelial populations are quite heterogeneous and are both vasculature type- and organ-specific. There are profound molecular, functional, and phenotypic differences between arterial, venular and capillary endothelial cells and endothelial cells in different organs. Given this endothelial cell population diversity, it has been challenging to determine the origin of endothelial cells responsible for the angiogenic expansion of the vasculature. Recent technical advances, such as precise cell fate mapping, time-lapse imaging, genome editing, and single-cell RNA sequencing, have shed new light on the role of venous endothelial cells in angiogenesis under both normal and pathological conditions. Emerging data indicate that venous endothelial cells are unique in their ability to serve as the primary source of endothelial cellular mass during both developmental and pathological angiogenesis. Here, we review recent studies that have improved our understanding of angiogenesis and suggest an updated model of this process. Cells that line the inside of veins possess a unique ability to grow new blood vessels and a better understanding of these cells could lead to new treatments for cancer, autoimmunity and other diseases associated with abnormal blood vessel formation. Michael Simons and colleagues from Yale University School of Medicine in New Haven, USA, review the attributes of venous endothelial cells, such as their unique ability to proliferate and migrate against blood flow, and then to form new intricate networks of minute blood vessels, in response to appropriate signals. The authors discuss emerging evidence implicating these cells in a variety of diseases, and suggest that drugs aimed at modulating the molecular function or migratory activities of venous endothelial cells could be used to correct abnormal blood vessel expansion. Endothelial cells can be categorized into several subtypes depending on the type of blood vessels they are found in and on their anatomic location. The three principal types of blood endothelial cells are arterial, venous, and capillary cells, and lymphatic endothelial cells form a distinctly different endothelial subpopulation4. In addition to these well-defined endothelial populations, there is also a population of endothelial tip cells. These cells are characterized by both their specific location at the front of the forming vascular network and the expression of specific genetic programs. Unlike arterial, venous, and capillary endothelial populations, the tip cell population is transient, and any endothelial cells at the vascular edge may assume tip cell features7.The term angiogenesis refers to the process of expansion of a preexisting vascular network. It requires the precise control and coordination of the proliferation, migration, and differentiation of endothelial cells (ECs)Because these different endothelial subtypes are exposed to distinct local microenvironments, including factors such as oxygen level, nutrients, and mechanical forces, from the flow of blood, it is not surprising that each endothelial subtype has a distinct gene expression pattern. Other factors, such as functional specialization  or anatomic location , also have a distinct genetic imprint. Given this endothelial diversity, the source of endothelial cells responsible for angiogenic vasculature expansion remains poorly defined.8 reported that venous endothelial cells are the primary source of endothelial cells responsible for angiogenic expansion both during embryonic development and in disease settings. Remarkably, venous endothelial cells are the only type of endothelial subtype capable of responding to hypoxia-induced angiogenic signaling by proliferating and migrating against the blood flow to form the new vascular bed. Abnormalities in this process account for pathological abnormalities, such as arteriovenous malformation (AVM) or excessive angiogenesis, that are seen in diabetic retinopathy.A recent studySeveral recent observations also suggest that the role of venous endothelial cells is conserved throughout different settings and species. Given that venous endothelial cells are the primary cellular source of angiogenesis, a better understanding of their biological features will help to facilitate the development of pro- and antiangiogenic therapies. In this review, we will provide a summary of recent advances in this field that focus on endothelial migration and the primary angiogenic role played by the venous endothelium.13. Thus, the hypoxic environment in a growing tumor or in the developing retina is seen as a source of the principle angiogenesis driver vascular endothelial growth factor A (VEGF-A). It is produced locally due to increased expression of hypoxia-inducible factor 1 alpha (HIF1α), which is a transcription factor regulating VEGF-A expression. This increased VEGF-A production stimulates the growth of new vasculature that, once formed, increases oxygen supply, relieves local ischemia, and arrests further vessel growth , angiogenic receptors , and others (DLL422 and ESM118).In this model, tip cells, a specialized but transient form of endothelial cells characterized by the expression of high levels of VEGF receptor 2 (VEGFR2) and the absence of a lumen, play a central role in the angiogenic response. VEGF binding to VEGFR2 on tip cells results in the activation of a number of tip endothelial cell-specific genes23. While tip endothelial cells guide the growing sprout, endothelial cells located just behind the tip cell position, known as stalk cells, form the body of the angiogenic sprout containing the blood vessel lumen. Similar to tip cells, stalk cells are also a transient endothelial cell subtype. In contrast to tip cells, stalk cells have fewer filopodia and reduced levels of VEGFR2. They support the extension of angiogenic sprouts, forming the lumen of the new vessel and maintaining a connection with the preexisting capillary bed27. Importantly, fate-mapping studies confirmed the contribution of tip cells to angiogenesis, demonstrating that a high proportion of the newly formed arterial endothelium is of tip cell origin29.This distinct gene expression profile enables tip endothelial cells to sense angiogenic cues and the VEGF gradient and guide newly formed angiogenic sprouts toward the hypoxic area31. Overall, endothelial NOTCH activation prevents excessive angiogenesis by reducing the overall number of tip endothelial cells. In the absence of this mechanism, such as in DLL4+/− mice23, an excessive number of new vessels are formed, resulting in defective tissue perfusion.To prevent a runaway train of angiogenesis, the process of tip vs. stalk cell specification and angiogenic sprouting is regulated by the activation of Notch signaling at endothelial cell–cell contact sites. An endothelial cell exposed to VEGF upregulates its expression of the Notch ligand Delta-like 4 (DLL4), thereby acquiring a tip cell fate. This newly minted tip cell interacts with NOTCH receptors on adjacent endothelial cells, resulting in NOTCH cleavage and the release of the intracellular Notch intracellular domain (NICD). NICD translocates into the nucleus, binds to RBPJ, and then activates NOTCH target genes. This converts endothelial cells with activated Notch signaling into stalk cells, limiting the number of newly formed sprouts. This promotes vascular quiescence, stabilizes EC-EC junctions, and suppresses proliferationOne shortcoming of the traditional tip/stalk specification-centered concept of angiogenesis discussed above Fig.  is that 33. While there are a number of enzymes capable of digesting different extracellular matrix components, matrix metalloproteinases (MMPs) are particularly important. Endothelial activation in response to VEGF and the influx and activation of proinflammatory cells, such as circulating and tissue-residing macrophages and T cells, leads to the local production of MMPs , the extent of which is positively correlated with the flow intensity40. Thus, NO induces the dilation of blood vessels, thereby lowering vascular resistance and facilitating tissue perfusion. In addition, flow-mediated shear stress regulates the expression of arterial and venous marker genes, including DLL441, EphrinB242, and KLF443, inflammatory markers , and vasodilation factors (COX2 and PGI246), among others. Endothelial cells under laminar flow are polarized and undergo extensive rearrangements of intracellular organelles47. One such key cytoskeletal rearrangement is the polarization of the Golgi apparatus48. This is important in the context of cell migration. The orientation of the Golgi complex is coupled to the direction of cell migration and can be used as an indicator of where the cell in question is going. This has been described in a number of cell types, including fibroblasts49, natural killer (NK) cells50, epithelial cells51, and cancer cells52. For example, a study of Golgi complex polarization in fibroblasts during “wound”-induced migration in vitro showed that cells located far from the wound exhibited random Golgi orientation. However, the Golgi in cells near the wound’s edge were polarized toward the wound, indicating the direction of their migration53.In addition to sensing VEGF gradients and responding to other circulating or locally present growth factors, endothelial cells are also exposed to dynamic forces generated by the flow of blood. The ability to sense and respond to these forces is a key feature of the endothelium and governs much of its behavior Fig. 38,39. La54.Likewise, endothelial cells under laminar flow display an aligned microfilament network and an upstream polarization of the Golgi Fig. . The Gol57, coronary artery endothelial cells58, and the aortic endothelium59. These findings suggest that it is a conserved feature among endothelial cells from different vascular beds. A study of endothelial Golgi-nucleus polarization under different magnitudes of fluid flow showed that in the majority of cells, the Golgi complex was polarized regardless of the flow magnitude.The upstream polarization of the Golgi towards the flow direction was also observed in human umbilical vein endothelial cells60 demonstrated the upstream polarization of endothelial cells in the dorsal aorta and arterial/venous intersegmental vessels. Endothelial cells in blood vessels with flow rapidly polarized once the flow started, while those without flow showed random polarization. These findings indicate that blood flow is the paramount factor determining the direction of endothelial polarization in vivo. In line with this observation, the upstream polarization of endothelial cells has been reported in a number of in vivo models and settings. In the postnatal retina vasculature, Golgi marker (Golph461 and GM13048) immunostaining showed upstream polarization in both arteries and veins. Endothelial cells in various mouse organs, including carotid arteries, jugular veins, thoracic aorta and the vena cava57, femoral62, and coronary arteries63, are also polarized against blood flow. Other species, including pigs (thoracic aorta and inferior vena cava)64 and rabbits (inferior vena cava)64, have also been reported to display the upstream polarization of endothelial cells against flow.In addition to these in vitro studies, recent studies using zebrafish clearly showed upstream polarization of endothelial cells in vivo. Using transgenic zebrafish expressing endothelial-specific nEGFP ) and endothelial-specific mCherry-labeled Golgi marker (Tg(fli1a:B4GALT1-mCherry), Kwon et al.65. Using live confocal imaging of a transgenic mouse embryo, Udan et al.65 demonstrated that endothelial cells in capillaries migrate against the bloodstream. They end up in large arteries and contribute to the expansion of the artery diameter. Moreover, reduced blood flow67 in Mlc2a−/− (myosin regulatory light chain 2) mice showed that the hemodynamic force is essential for the reverse migration of endothelial cells and their recruitment into arteries65. Reverse migration has also been reported using a fin regeneration model in a transgenic zebrafish line carrying reporter genes for endothelial nuclei (Tg(fli1a:nEGFP)y7) and arterial-EC membranes (Tg(–0.8flt1:RFP)hu5333)68. Time-lapse confocal imaging over 24 h demonstrated that venous endothelial cells migrated against the bloodstream, took over tip cell positions, and then continued the reverse migration toward the arterial bed. Likewise, endothelial cells in mammals also showed similar migration patterns. Time-course lineage tracing with tip cell-specific inducible Cre recombinase-expressing mice (Esm1(BAC)CreERT2; R26mTmG) demonstrated that tip cells in postnatal retina vasculature are recruited into arteries but not veins, supporting the reverse migration paradigm68. The most recent study reported that the reverse migration of the endothelium is a conserved feature among different organs, including the brain, retina and dermis, using a venous endothelium-specific inducible Cre line (Gm5127(BAC)CreERT2;R26mTmG)8. Lee et al.8 found a novel venous endothelium marker and generated venous EC-specific inducible Cre and Dre driver mouse lines. Time-course lineage tracing revealed that venous endothelial cells migrate against the bloodstream and sequentially differentiate into capillary, tip, and arterial endothelial cells8. Strikingly, the majority of endothelial cells (~80%) in the postnatal angiogenic stage originate from the venous endothelium, indicating that venous endothelial cells are the primary source of EC during angiogenic expansion  and nuclei (Flk1-H2B::eYFP)69. This implies that highly proliferating venous endothelial cells migrate against the bloodstream, differentiate into other endothelial subtypes, and serve as the primary source of endothelial cells for the expansion of the vascular network  induces the reverse migration of venous endothelial cells with mitotic activity in adult zebrafish68. This demonstrates that quiescent venous endothelial cells can reinitiate proliferation and reverse migration in response to pathological stimuli.Venous endothelial cells are more mitotically active than those in other vascular bedsork Fig. . Venous 72 , a coreceptor of TGFβ/BMP signaling, alters endothelial shapes under blood flow78, and vessels with ENG loss developed AV shunts in both zebrafish78 and mice75. Activin receptor-like kinase 1 (Alk1)-deficient endothelial cells also display polarization defects under flow56 that also play a role in the development of AVM79. SMAD4, a downstream transcription factor of the TGFβ/BMP signaling pathway, is yet another gene involved in the control of endothelial alignment. While normal endothelial cells are polarized and aligned along the axis of the flow, SMAD4-deficient endothelial cells exhibit a lack of alignment and reduced polarization58. This observation has also been confirmed in the retinal vasculature in in vivo settings. Misdirected endothelial alignment and polarization result in AVM development in endothelium-specific SMAD4 KO mice (SMAD4iECKO)77. While the venous endothelial Golgi complex is polarized toward the retinal periphery in normal mice, those in SMAD4iECKO mice polarized toward arteries displaying venous endothelial cells. Thus, in SMAD4iECKO mice, the polarization is abnormally directed  are one of the most widely used models to investigate mechanistic details of endothelial dysfunction in angiogenesis because the mouse AVM model provides a highly detailed view of the AVM structure in the superficial layer of the retinal vasculature. Arteriovenous malformations are a common vascular disorder characterized by an abnormal connection between an artery and a vein (AV shunt). They are caused by loss-of-function mutations of genes in the TGFβ/BMP signaling pathway, including ALK175 and venous-capillary marker (Endomucin)80 but not arterial markers (Jag1 and Aplnr) are highly expressed in AV shunt lesions of ENGiECKO mice. AVMs in SMAD4iECKO and ALK1iECKO mice also show upregulated expression of venous endothelial markers, including EphrinB2 and Nrp277. Interestingly, deletion of ENG with venous/capillary endothelium-specific Cre (Apj-CreERT2) and pan endothelium-specific Cre (Cdh5-CreERT2) led to a similar incidence and frequency of AVMs, indicating that arterial ENG does not protect AVM development. Likewise, deletion of ALK1 with venous/capillary endothelium-specific Cre (Mfsd2a-CreERT2) leads to AVM development but not tip- (Esm1-CreERT2) or arterial EC (Bmx-CreERT2)-specific deletion56. These in vivo findings imply that endothelial cells in capillaries and/or veins are the main drivers of the development of AVMs.Growing evidence indicates that venous endothelial cells also play a crucial role in the development of AVMs. For instance, endothelial cells in AV shunt lesions are known to have a venous identity. Venous endothelium marker (EphrinB2)SMAD4fx/fx;Cdh5-CreERT2;Gm5127-DreERT2; Rosa26RC::RG), Lee et al.8 traced the origin of endothelial cells in AVM lesions to the venous endothelium. Time-course analysis of AVMs showed that AVM formation is initiated by abnormal sprouting from veins and that the majority of endothelial cells in AVMs originate from venous endothelial cells8. The lack of polarization/alignment in SMAD4-deficient endothelial cells against flow58 and the excessive proliferation of endothelial cells in AVM lesions8 suggest that AVMs are consequences of the undirected migration of excessively proliferating venous endothelial cells. Recent single-cell RNA sequencing analysis of endothelial cells in a cerebral cavernous malformation (CCM) model also revealed that those in CCM lesions originate from a subset of venous/capillary endothelial cells81, indicating that the role of venous endothelial cells in malformation is not limited to AVMs. Jin et al.82 reported that the mosaic loss of ENG (by low-dose tamoxifen injection in ENGiECKO mice) resulted in initial AVM formation in arterioles. Identification of the origin of ECs in those lesions using EC subtype-specific reporter lines may explain the discrepancy in the ENG-deficient setting.Using fate mapping that employed a dual Cre and Dre recombinase system that deletes SMAD4 in the pan endothelium and simultaneously labels venous endothelial cells by tamoxifen administration  conditions for several days leads to the formation of a vaso-obliteration (VO) zone, and returning them to the room air triggers neovascularization to alleviate the VO zone  model has been widely used in cardiovascular research. While it closely resembles retinopathy of prematurity90. Zebrafish have been extensively utilized as an in vivo model of angiogenesis during wound healing for over a decade because of their ability to regenerate caudal fins after amputation91. Major players involved in the angiogenic process  are conserved between species and function similarly93. The availability of various genetically modified zebrafish lines expressing fluorescent proteins in each endothelial subtype  made this model more attractive for the intravital imaging of postinjury angiogenesis99. Amputation of the caudal fin activates endothelial cells in preexisting vascular beds to invade the wound area and establish a new vascular network  is also highly expressed in the tumor endothelium, implying the importance of the venous endothelium in tumor angiogenesis101.w are still largely unexplored. Recent advances in scRNA-sequencing technology have enabled large-scale sequencing and analysis of various endothelial cell populations in cancer. Interestingly, studies of the tumor endothelium have revealed that the numbers of activated postcapillary venous endothelial cells were increased in the lung tissues of cancer patients compared to healthy controls103. Clearly, alternative strategies are needed to overcome the current limitations. To date, most efforts to control the process of angiogenic vascular growth have mainly focused on VEGF-A and its receptors, while other factors regulating this process have been largely ignored. Recently, a growing body of evidence has started indicating the importance of venous endothelial cells in developmental and pathological angiogenesis in a number of pathological settings. Venous endothelial cells are distinct from other endothelial subtypes in that they possess high mitotic activity, are able to migrate against blood flow, and can assume the molecular and functional identity of other endothelial subtypes. Despite much progress in understanding the role of the venous endothelium in various developmental and pathological settings, elucidation of the molecular mechanisms that confer these features on the venous endothelium has only just begun. One important feature of this endothelial subtype is that quiescent venous endothelium, unlike other endothelial subtypes, can be reprogrammed to activate their ability to migrate in response to angiogenic and mechanical stimuli. This implies the presence of specific molecular control mechanisms that regulate the reactivation process and migratory activity in the venous endothelium. A better understanding of these mechanisms and why they are unique to the venous endothelium may enable us to develop better therapeutic strategies for angiogenesis-related diseases.Despite three decades of development, the success of pro- and anti-angiogenic therapies has been rather modest. The best results achieved in retinal diseases were from the use of anti-VEGF therapies that resulted in the significant preservation and improvement of vision


To date the true global incidence of intrahepatic cholangiocarcinoma (iCCA) and the underlying risk factors remain to be fully defined, in particular, the role of occupational and environmental factors. Currently, the putative role of asbestos exposure as a risk factor for iCCA is gaining increased attention in the international scientific community and agencies.In this commentary we review and integrate available epidemiological and mechanistic evidences that support a potential role of asbestos exposure in iCCA etiology. Intrahepatic cholangiocarcinoma (iCCA) arises from the epithelial cells within the liver bile ducts and represents the second most common primary liver cancer after hepatocellular carcinoma (HCC). One of the major challenges in the field of iCCA is to define the true global incidence of this disease and the underlying risk factors. Although still considered a rare malignancy in Europe and USA, in the past decades age-standardized iCCA incidence appeared to steadily increase in most locations worldwide . However2endemic, parasitic infections with Clonorchis sinensis and Opisthorchis viverrini represent the dominant risk factor for this disease [Currently certain pathological/genetic conditions  have been recognized as risk factors for iCCA 1. In East disease 7. Intere disease . HoweverIn this commentary we review current epidemiological and molecular evidence that support a potential link between asbestos exposure and increased iCCA risk.The possible association between asbestos exposure and biliary tract cancers was firstly observed some decades ago by Selikoff et al. who reported a significant increase  of death for gall bladder/bile duct cancer among 17 800 asbestos insulation workers of USA and Canada from 1967 to 1986 11, even 12In order to hypothesize a role of asbestos exposure in iCCA development, the detection of fibers in the intrahepatic biliary tract is mandatory. Two recent exploratory studies reported the deposition of asbestos fibers in the bile/gallbladder of patients with benign diseases of the biliary tract and, more interestingly, in the liver of iCCA patients living in Casale Monferrato, an area of Italy at high level of environmental exposure to asbestos 15. These16From a molecular point of view, the knowledge of the mechanisms driving iCCA carcinogenesis is rapidly evolving due to the availability of high throughput analytical technologies such as next-generation sequencing. In lung cancer, genomic profiling identified a distinctive molecular signature in asbestos-exposed patients compared to not-exposed, including copy number aberrations in the 2p16, 9q33.1 and 19p13 loci and MRPL1, INPP4A, SDK and SEMA5B somatic mutations 19. In mali2127Currently, about 125 million of people are still environmentally exposed to asbestos worldwide, even in countries that banned its use . Recentl30Overall, the body of evidences coming from epidemiological and mechanistic studies addresses to a putative causal role of asbestos in the genesis of iCCA, deserving further investigations in large observational studies with accurate asbestos exposure assessment.


Communication between microglia and other cells has recently been at the forefront of research in central nervous system (CNS) disease. In this review, we provide an overview of the neuroinflammation mediated by microglia, highlight recent studies of crosstalk between microglia and CNS resident and infiltrating cells in the context of ischemic stroke (IS), and discuss how these interactions affect the course of IS. The in-depth exploration of microglia-intercellular communication will be beneficial for therapeutic tools development and clinical translation for stroke control. Microglia are initially termed “the third element of center nerve” and discovered as resident macrophages in the central nervous system (CNS). Microglia are primarily responsible for innate immunity in the brain and play a pivotal role in maintaining the homeostasis of the CNS through immune surveillance. As such, microglia can rapidly respond to the local milieu and drive acute inflammation as the defense and repair mechanism to maintain brain homeostasis. Although they are involved in immune responses within the CNS, they possess many additional capabilities beyond simple immune surveillance. Microglia, by themselves or interacting with other glial cells (astrocytes and oligodendrocytes), can instruct neurogenesis, regulate neural function, facilitate synapse formation and preserve the integrity of the blood-brain barrier  is one of the leading causes of mortality and disability worldwide, and it is primarily caused by thromboembolic occlusion of a major artery that supplies the brain  and emphasizes the importance of the dynamic interactions of endothelial cells, astrocytes, microglia, and neurons. Microglia, the pivotal immune sentinels, interact with other components in the NUV to form a complex network involved in the progression of IS .In addition to their pivotal role as immune sentinels of the brain, emerging data suggest that microglia are critical to neuronal homeostasis. Meanwhile, neuronal activity is confirmed to be a driver of microglial action through “on” and “off ” signals. As such, most studies focus on the mechanisms of bidirectional microglia-neuron communication and demonstrate that there are a lot of cell-cell signals involved in indirect interaction  mediate microglia-neuron communication  Damaged neurons are not merely passive targets of microglia but rather regulate microglial activity through different signals. CX3CL1/CX3CR1 are the most studied ligands and receptors pairs involved in controlling microglial activation. CX3CL1, which mainly derives from neurons, interacts with CX3CL1R expressed in microglia and directly induced microglial migration, proliferation, and even the ability to eliminate the degenerated neuron debris. CX3CL1 released from injured neurons may signal microglia as a “help me” signal -dependent phagocytosis of apoptotic neurons  and NF-κB/p65 , the unique cell type implied in CNS myelination, are sensible to ischemic damage. Swelling of OLs occurred 30 min after arterial occlusion, and a large number of OLs died 3 h after ischemia, which appeared earlier than neuronal death in the ischemic area  disruption is a major pathological hallmark in the course of IS  claudin-5 to the endothelium and contact EC to maintain BBB integrity. While sustained inflammation triggers activated microglia to impair BBB function by engulfing astrocytic end-feet  after IS  term enrichment analysis for microglia differentially expressed genes (DEGs) suggested that the genes associated with microglial activation were overexpressed, while the genes involved in homeostatic cellular metabolism were downregulated. In addition, macrophage depletion reduced the microglia activation in both the ipsilateral cortex and striatum, thereby facilitating neurological recovery after MCAO , monocyte infiltration decreased in As mentioned above, microglia rapidly activate after IS onset and promote the invasion of peripheral immune cells into the brain by cytokines and chemokines , CD4+ T helper cells (Th), and regulatory T cells  were also found within the ischemic penumbra. The infiltrating double-negative T cells could promote M1-MG transition through FasL/PTPN2/TNF-α signaling pathway and thus aggravate ischemic brain injury , a subset of T cells characterized by the transcription factor Foxp3, account for 5%–10% of the peripheral CD4In conclusion, these findings show that Th2/ Treg cells exert a neuroprotective role related to reducing the inflammatory response and promoting the M2-MG response.Minimal amounts of B cells can normally be detected in the brain but are transported in large quantities to CNS during injury or disease  are critical components of the innate immune and adaptive immune response. DCs detect and accumulate foreign antigens and present antigens to naive T cells , lipid-droplet-accumulating microglia (LDAM), proliferative zone associated microglia (PAM), and glioma-associated microglia (GAM) states  from animal models to humans is yet to yield a positive result to date , the Tianjin Natural Science Foundation (No. 19JCZDJC37100), and the School of Integrative Medicine Foundation in Tianjin University of Traditional Chinese Medicine (No. ZXYKYQDLX202001).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.