SLC6A1 pathogenic variants have been associated with epilepsy and neurodevelopmental disorders. The clinical phenotype includes different seizure types, intellectual disability, and psychiatric symptoms affecting mood and behavior. Few data regarding neuropsychological features have been described, and details on cognitive profiles are often missing due to the lack of standardized tests.SLC6A1. We also collected data on epileptic features, EEGs, and brain MRIs. Additionally, we reviewed neuropsychological data from 204 previously reported patients with SLC6A1 pathogenic variants.We retrospectively reviewed the neuropsychological assessments of five subjects carrying heterozygous missense genetic variants in SLC6A1 pathogenic variants have mild-to-moderate intellectual disability and that one-third of cases have autistic traits.In our series, at the last evaluation (median 12.6 years), three patients had borderline intellectual functioning, one patient had mild cognitive impairment, and one patient presented with a moderate cognitive disability. Three out of five patients underwent at least two neuropsychological evaluations, which revealed a worsening of cognitive functions over time. We detected attention deficits in all patients. In addition, we observed anxiety, disruptive behavior disorder, emotional instability, and hetero aggressiveness. We also performed a literature review that highlighted that most of the patients with SLC6A1-related epilepsy mostly present with mild-to-moderate intellectual disability, often associated with attention disorders. Such symptoms may worsen over time. Periodic standardized neuropsychological tests may be useful tools to follow development over time, and patient-specific rehabilitation programs could be tailored consistently.Based on the literature review and the detailed description of our cases, we conclude that patients with  SLC6A1 [MIM: 137165] is located on the short arm of chromosome 3 (3p25.3) and encodes sodium chloride-dependent GABA transporter type 1 (GAT-1), a voltage-dependent transmembrane protein (Solute carrier (SLC) proteins are ubiquitous transmembrane transporters with a crucial role in maintaining the physiological functions of organisms and as medication targets . SLC6A1  protein . GAT-1 a protein .SLC6A1 destabilize the global protein conformation, causing a loss of GAT-1 function that leads to alterations in GABA levels in the synaptic cleft   ( (EMAtS) . Later,  (EMAtS) . Neverthsyndrome . Overalles (TCS) .SLC6A1-related epilepsy , which includes 204 previously published patients with SLC6A1 pathogenic variants.In this study, we describe the neuropsychological profile of five new patients carrying SLC6A1 pathogenic variants who were followed at Bambino Gesù Children’s Hospital in Rome, Italy. SLC6A1 pathogenic variants were identified in all patients through a trio-based New Generation Sequencing (NGS) epilepsy gene panel. We collected clinical data, including sex, family history, age at seizure onset, seizure semiology and frequency, intellectual disability, behavioral and psychiatric disorders, EEG and brain MRI.We retrospectively reviewed the medical charts of five patients with Seizure types and epilepsy syndromes were classified according to the Commission on Classification and Terminology of the International League Against Epilepsy .We retrospectively reviewed all cognitive and behavioral evaluations for all patients performed with the following scales:Developmental and cognitive assessment:Griffiths Mental Development Scales (GMDS).- Wechsler Intelligence Scale for Children, fourth edition (WISC IV).- Wechsler Adult Intelligence Scale, fourth edition (WAIS-IV).- Wechsler Preschool and Primary Scale of Intelligence, third edition (WPPSI-III).- Raven’s Progressive Matrix (RPM): PM38 and PM47.- Leiter- R- Behavioral assessment:Child Behavior Checklist (CBCL) which includes eight syndrome scales  that lead to group subjects into two behavioral areas  and resulting in a total score.- Conners’ Parent Rating Scale-Revised: Long Form (CPRS-R:L).- This study was performed according to ethical principles for medical research involving human subjects stated in the Declaration of Helsinki and was approved by the local ethics committee.SLC6A1 pathogenic variants. We identified a total of 204 patients carrying SLC6A1 genetic variants. The search strategy included the terms “SLC6A1” and/or “Epilepsy” on PubMed. References were also identified manually from relevant articles and by searching through the authors’ files. We limited the review to 50 English-language articles. We excluded patients with microdeletions encompassing SLC6A1 and extended the search to other genes that could be causative of misleading data.We also reviewed the literature from January 2012 to February 2023 related to all published patients with For all reported patients, we collected the genetic variants  and clinical data . This is a 25-year-old male with normal psychomotor development before seizure onset. Epilepsy started at the age of 2 years and 3 months with atonic seizures, followed by absences with eyelid myoclonia, myoclonic-atonic, and myoclonic seizures. He was treated with valproate (VPA), ethosuximide (ETS), and clonazepam (CNZ). The last seizure was at the age of 3 years and 7 months; therefore, in adolescence, anti-seizure medications (ASMs) were discontinued without seizure relapse.At the age of 7 years, the Leiter-R scale, exploring the nonverbal cognitive profile, revealed a borderline cognitive level (IQ = 80). At the age of 12 years, we performed a second cognitive evaluation, which detected mild cognitive impairment (IQ = 67). He attended school with a support teacher. He also had a behavioral disorder, including attention deficit and stereotyped behavior. At the age of 25 years, at the last follow-up (FU), he had mild ID requiring support in personal autonomy and self-care.This is an 18-year-old male. Epilepsy started at the age of 6 years with episodes of absences with and without eyelid myoclonia. At the age of 10 years, he presented with tonic seizures, and by the age of 12, he started to present also with atonic seizures. He was treated with several ASMs, such as VPA, ETS, carbamazepine (CBZ), and clobazam (CLB), with poor efficacy. Tonic and atonic seizures have been controlled with ASMs since the age of 15 years, while daily absences persist at the last FU.Independent gait was reached at the age of 18 months. He had speech delay: first words at 24 months; therefore, speech therapy was started.The first neuropsychological evaluation was performed at the age of 13 years using the nonverbal cognitive test PM 38, which detected borderline intellectual functioning in terms of fluid reasoning (IQ = 74). In addition, the CBCL showed several difficulties in attention, anxiety, and social domains. He also had difficulties in managing his emotional states, which often led to behavioral dysregulation. A rehabilitation program was indicated.We re-evaluated his cognitive performance at the age of 16 years with the PM 38. We found cognitive regression (IQ = 62), indicating mild cognitive delay. He needed school support and a differentiated school program.Finally, we evaluated the patient at the age of 18 years with the WAIS-IV, which showed a moderate cognitive delay (IQ = 44), with greater impairment in the working memory index. He also presented with anxiety, relational disorders, and depressive features, which may have affected cognitive assessments.This is a 6-year-old girl born at 35 weeks of gestation from a twin cesarean section. Independent walking was reached at the age of 16 months. Parents reported speech difficulties since the second year of life. Epilepsy onset was at the age of 3 years with atonic seizures and episodes characterized by psychomotor arrest and oral automatisms. Seizures were treated with CBZ, which worsened atonic seizures; after the replacement of CBZ with VPA and CLB, she reached complete remission of seizures.The first neuropsychological evaluation was performed at the age of 3 years and 2 months with GMDS, which showed a developmental age of 32.5 months versus a chronological age of 37 months (DQ = 88): lower scores were seen in language and oculomotor coordination.A second evaluation was performed at the age of 4 years and 4 months. The PM47 nonverbal cognitive scale showed an IQ of 90, with weakness in the areas of attention skills; this was not confirmed by the CPRS-R:L.Finally, we evaluated the patient at the age of 5 years and 9 months with the WPPSI III scale, which showed borderline intellectual functioning (IQ = 77), with greater impairment in the processing speed index. We observed a deficit in attentional skills, with worsening of both selective and sustained attention.She had been treated with psychomotor and speech therapies since the age of 3 years. Currently, the child attends primary school with a support teacher.This is a 6 years and 7 months old girl born from cesarean section after a normal pregnancy. At the age of 6 years and 5 months, she presented with epileptic seizures: TCS and absences with eyelid myoclonia were reported. She was treated with levetiracetam (LEV), lamotrigine (LTG) and CNZ with poor efficacy, while VPA led to significant seizure reduction.Motor development was normal in the first few years of life, except for delayed speech (first words at the age of 3 years) and phono-articulatory difficulties. At the age of 6 years and 5 months, she was assessed with the PM47 nonverbal cognitive scale (language barrier was clear), and she had an IQ of 70. At the same age, she also presented with behavioral disturbances characterized by oppositionality, emotional instability, heteroaggressiveness, attention deficit, and impulsivity. Poor frustration tolerance and fatigability were observed during the evaluation. Moreover, the use of CNZ had a negative impact on attention and behavior, inducing irritability, which was restored with drug withdrawal.The child is not currently undergoing rehabilitation treatment due to poor familial psychosocial conditions.This is a 6-year- and 6-month-old boy born after a normal pregnancy. His mother suffered from generalized epilepsy with absences since the age of 6 years. Neuropsychological development was normal during infancy. Epilepsy started at the age of 3 years with atonic seizures and absences with eyelid myoclonia successfully treated with VPA.The WISC IV at the age of 6 years and 6 months revealed a borderline cognitive level (IQ = 80) with strengths in processing speed and visual spatial index and weaknesses in working memory index. In addition, we detected a mild language delay. Over time, he also developed behavioral disturbances such as attention and executive function deficits, mild anxiety, and depression.de novo heterozygous missense variant , and two patients carried a maternally inherited genetic variant  in the SLC6A1 gene (Three patients carried a 6A1 gene . Patient6A1 gene . Three g6A1 gene .SLC6A1 pathogenic variants. Sex was reported in 140/204 (68.6%) patients: 75 (53.6%) were females.We collected 204 patients with SCL6A1 pathogenic variants were characterized as missense in 161/204 (79%), nonsense in 13/204 (6.3%), frameshift in 13/204 (6.3%), splice site in 10/204 (5%), inframe deletions in 5/204 (2.4%), and copy number variants (CNV) in 2/204 (1%) patients. Inheritance was available in 118/204 patients: 98/118 (83%) had a de novo genetic variant, and 20/118 (17%) inherited the genetic variant from parents.Epilepsy was reported in 169 subjects. Data on seizure types were available in 116/169 patients. Different seizure types were reported: absences with or without eyelid myoclonia (94/116), atonic (70/116), myoclonic (58/116), tonic–clonic (22/116), and focal (9/116) seizures, often coexisting in the same patient. Syndromic classification was possible for 64 out of 169 patients: EMAtS was reported in 48 patients, childhood absence epilepsy (CAE) in 15 patients and LGS in 1 patient.Neuropsychological assessments were sparsely reported in the literature, although data were often neither detailed nor standardized.Data on neurodevelopment were available in 119/204 patients (58.3%). Developmental regression was seen in 15/119 (12.6%) cases, sometimes evolving into DD or ID. DD or ID was reported in 98/119 (92.3%) patients, subclassified as mild (27), moderate (24), or severe (8).Eleven out of 110 (10%) subjects had normal IQ, and 1/110 (0.9%) had borderline intellectual functioning. In addition, learning disorders were mentioned in 5/11 patients with normal IQ.Regarding psychiatric features, ADHD was reported in 20 patients, ASD or autistic features in 64 patients, and behavioral disorders in 41 patients.SLC6A1 pathogenic variants includes epilepsy with various seizure types , neurodevelopmental disorders , and psychiatric features affecting mood and behavior . To date, few data on neuropsychological features have been reported; however, data on cognition and behavioral disturbances are often missing because of a lack of standardized testing.The clinical phenotype associated with Developmental delay has been frequently reported by parents even before epilepsy onset, as in three out of five of our patients . Once epData from the literature review report developmental regression in approximately 13% of patients around the age of 3 years . The disAdditionally, the severity of ID was also seldom detailed in the literature, and when reported, it ranged from mild to moderate in most cases.In general, no significant differences, in terms of cognitive impairment, were reported as related to seizure control . AccordiSLC6A1 genetic variants encompasses wide phenotypic variability, from epilepsy with or without ID to the DEE phenotype.Eleven patients with normal IQ were reported in the literature, even if learning disorders were observed in five of them. Six out of 11 patients with normal IQ had epilepsy and interictal epileptiform discharges at EEGs: these findings support that etiology, rather than epilepsy, has a major role in this condition . Most ofOur patients received several cognitive and behavioral tests. At the last neuropsychological assessment, three patients had borderline intellectual functioning, one had mild cognitive impairment, and the last patient had moderate cognitive disability. Three out of five had a worsening of cognitive functions over time. Patient 1 moved from borderline functioning to mild cognitive impairment, patient 2 from borderline to moderate cognitive delay, and patient 3 from normal to borderline functioning over 2 years without developing a clear-cut cognitive disability. What was seen in most patients was a delay in acquiring new skills rather than a clear regression. Adult patients achieved personal independence in daily activities, even if they live with their parents. They do not have jobs, and one of them still attends school with a support teacher and an individualized learning plan. They do not practice any rehabilitative or psychotherapeutic therapy. They achieved personal independence in daily activities with minimal supervision.These findings underline the importance of longitudinal assessments in patients with epilepsy over time, especially in children of developing ages. Accordingly, periodic cognitive evaluations with standardized testing, jointly with structured behavioral assessments, might be useful to adapt over time for a patient-specific rehabilitation program to identify the best cognitive functioning and reach good daily life autonomies during adulthood.Regarding behavioral disorders, data from the literature may be misleading. Few authors have properly analyzed these features, reported as aggressive or oppositional behaviors with psychiatric traits .Consistent with literature data , we founSLC6A1 patients with autism, from traits to ASD, are frequently related to developmental regression  can be found in the article/The studies involving human participants were reviewed and approved by the Ethic Committee of Bambino Gesù Children’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.MT, AB, AD, and CC collected and analyzed clinical data. AD and AN provided and analyzed genetic data. CQ and SC collected and analyzed neuropsychological data. MT, AB, and CQ wrote the first draft of the manuscript. SC, NS, FV, and AN supervised the study and reviewed the last version of the manuscript. All authors read and approved the submitted version.This work was supported by #NEXTGENERATIONEU (NGEU) and funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006)—a multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022). This work was supported by the Italian Ministry of Health with Current Research funds.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 effect of multidrug immunosuppressive protocols on the salivary glands is still unknown. This study aimed to determine the influence of immunosuppressive regimens based on calcineurin inhibitors (CNIs) and conversion to rapamycin on the morphology, apoptosis, and proliferation of rat salivary glands.Male rats received cyclosporin A (CsA), tacrolimus (FK-506), mycophenolate mofetil (MMF), rapamycin (Rapa), and prednisone (Pre) according to three-drug protocols: CMP , CMP/R , TMP , and TMP/R . Morphological and immunohistochemical and quantitative analyses of the salivary glands were performed.Structural changes in salivary glands were observed in all experimental groups, especially in the submandibular gland. In the salivary glands, the percentages of collagen fibers and TUNEL-, Ki67- and PCNA-positive cells were higher in the experimental groups vs. the control but were lower in the CMP/R and TMP/R groups vs. the CMP and TMP groups, with the exception of collagen fibers in the parotid gland in the TMP/R group vs. the TMP group.Long-term administration of CNIs in triple regimens and after conversion to rapamycin monotherapy, causes morphological changes in the salivary glands of rats. Immunosuppressive treatment based on CNIs is associated with an increase in collagen accumulation. The effects of the conversion of treatment with CNIs to rapamycin in immunosuppressive protocols in rat salivary glands lead to decreased fibrosis, apoptosis, and proliferation. These changes may possibly prevent abnormalities resulting from the application of CNIs.The online version contains supplementary material available at 10.1007/s43440-023-00521-0. Long-term use of immunosuppressive drugs is common in vascularized organ transplant patients, as well as in inflammatory and autoimmune diseases. In transplant recipients, these drugs are used to prevent immune-borne rejection of transplanted organs, which is associated with increased patient survival. Transplant rejection is a complex process influenced by both immunological and nonimmunological factors. It is important to choose the proper immunosuppressive treatment protocol, which will have a low rate of side effects and significantly improve the quality of life of patients –3.In transplant recipients, the standard triple medication protocol includes a combination of calcineurin inhibitors (CNIs), including cyclosporine A (CsA) and tacrolimus (FK-506), an antiproliferative agent, e.g., mycophenolate mofetil (MMF), a corticosteroid, e.g., prednisone, and less mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin (sirolimus) . The useCNI-based immunosuppressive treatment protocols are important in maintaining the proper function of the graft, but they have a narrow therapeutic window and show a number of side effects. The long-term use of CNIs is associated with nephrotoxicity that may contribute to chronic graft dysfunction. However, it has been shown that rapamycin, showing strong immunosuppressive and antiproliferative effects and high efficiency in preventing acute rejection of transplants, may act synergistically with CsA, inhibiting the proliferation of activated lymphocytes. Moreover, rapamycin has a favorable nephrotoxicity profile compared to CNIs. It has also been suggested that multidrug therapy with rapamycin may be associated with delayed drug resistance –6. It isThe available literature provides information on the known side effects of CsA and FK-506 in the form of gingival overgrowth and alveolar bone loss , 11. In There are very few scientific reports on the effect of immunosuppressive drugs on the morphology of salivary glands –18. Unfon = 6 per cage), had free access to water throughout the experiment and received a high-quality specialized laboratory diet of LSM type . The housing temperature was maintained at a humidity of 50 ± 5% and 21 °C, and the dark:light cycle was set at 12-h light:12-h dark. The initial body mass of the animals was approximately 305 g.The experiment was conducted on 30 3-month-old male Wistar rats. The sexually mature animals were obtained from a licensed breeder from the Institute of Occupational Medicine  and had certificates issued by an official veterinarian confirming their health and genetic status. The rats were adapted to the experiment for 2 weeks. The animals were typically housed in simple cages consisting of one open space (n = 6 in each group): the control group and four experimental groups . The rats in the control group were administered just bread balls vehicle with no added drugs. In the experimental groups rats received orally the following immunosuppressive drugs: CsA , FK-506 , MMF , prednisone , and rapamycin , according to the three-drug protocols used in patients after organ transplantation. The following daily drug doses were used: CsA—5.0 mg/kg of body weight, FK-506—4.0 mg/kg of body weight, MMF—20.0 mg/kg of body weight, prednisone—4.0 mg/kg of body weight, and rapamycin—0.5 mg/kg of body weight. In the CMP group, rats received CsA, MMF, and prednisone; in the CMP/R group, rats received CsA, MMF, and prednisone in the first 3 months and rapamycin in the last 3 months; in the TMP group, rats received FK-506, MMF, and prednisone; and in the TMP/R group, rats received FK-506, MMF, and prednisone in the first 3 months and rapamycin in the last 3 months. The animals received drugs using their pharmaceutical form in bread balls every day for 6 months. To obtain reliable results, the drug doses were adjusted to the body mass of rats and properly calculated considering the metabolic differences between rats and humans. The drug concentrations in the blood of the rats were within the therapeutic range as previously published by Grabowska et al. [The animals were randomly divided into five groups .Rats were anesthetized intraperitoneally with 50 mg/kg ketamine hydrochloride. During sectioning, the parotid, submandibular and sublingual salivary glands of rats were obtained. Salivary glands of the control and experimental groups were fixed in 4% paraformaldehyde in phosphate buffer for 24 h at 4 °C and embedded into paraffin. The paraffin blocks were cut into 3-μm sections, which were placed on polylysine-coated slides for further analysis.Sections of the parotid, submandibular, and sublingual salivary glands were deparaffinized using xylene followed by hydration in a series of graded alcohols. Next, the sections were stained using standard methods: hematoxylin and eosin (HE) for routine histological examination and Masson trichrome to reveal the collagen fibers.Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was carried out according to the manufacturer’s instructions  previously described in detail by Grabowska et al. . The negThe immunostaining procedure was performed according to the protocol previously described in detail by Grabowska et al. . To reveMasson trichrome-stained and also TUNEL-, Ki67-, and PCNA-immunostained slides were scanned at a magnification of 400 × on the Aperio AT2 digital slide scanner . The scanning resolution was 0.25 μm/pixel. The background illumination levels were calibrated using a prescan procedure. Moreover, the scanner was configured to minimize focus problems. The obtained digital slides of the tissues were examined on the computer screen using the ImageScope viewer .For the quantitative analysis of collagen fibers in rat salivary glands stained with Masson trichrome, a positive pixel count v9 algorithm  was used. Other parameters were set to achieve compliance with the visual evaluation. The areas of analysis were manually determined. The percentage of collagen fibers that were positive for Masson's trichrome staining was calculated in 30 fields in each group.n = 6) with an average analysis area not less than 0,96 mm2).For the quantitative analysis of TUNEL-, Ki67-, and PCNA-positive cells in the salivary glands, a nuclear v9 algorithm  was used. Other parameters were set to achieve compliance with the visual evaluation, taking into account the threshold for a positive result, a brown color of the reaction in the cell nucleus. The areas of analysis were also manually determined. Using the algorithm, the percentage of positive nuclei was determined. The total number of positive nuclei was counted in 30 fields in each group (five high-power fields from each rat (n = 6), the non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test for post hoc analysis was used to assess the differences between the groups. The probability p ≤ 0.05 was considered statistically significant.All statistical analyzes were performed by using TIBCO Statistica version 13.3 . The medians and ranges were calculated. Since the obtained values failed the normal distribution assumption and the groups of animals were small  and interlobular excretory ducts were lined with columnar epithelial cells. Moreover, in the parotid gland, adipose tissue often occurred Fig. E. IntersIn rat submandibular glands from the control and experimental groups, typical tubuloalveolar mixed protein-secreting serous acini and mucin-secreting mucous acini with a predominance of serous acini were noted. Very few scattered mucous acini within the main serous portion of the gland were observed. The acinar cells were mainly basophilic. The mucous acini were characterized by polyhedral cells with flattened and basally located nuclei. The serous acini were similar to those in the parotid gland. The granular ducts, characteristic of rodents, were lined by cuboidal or columnar epithelial cells with eosinophilic secretory granules in the cytoplasm. The intercalated ducts were lined by cuboidal epithelial cells, while striated ducts and interlobular excretory ducts were lined with columnar epithelial cells with nuclei located in the central or apical region. It is worth mentioning that within the submandibular gland, no adipose tissue was noticed compared to the parotid salivary gland Fig. H, J. In In the rat sublingual glands from the control and experimental groups, normal tubuloalveolar mixed protein-secreting serous acini with a clear predominance of mucin-secreting mucous acini were found. In most terminal secretory units, typical mucous acini surrounded by a few serous acini forming serous demilunes were observed. The intercalated ducts were lined by cuboidal epithelium, whereas the interlobular excretory ducts were lined with columnar epithelial cells. In addition, within the sublingual gland, no adipose tissue was observed  = 70.63; p = 0.002 and p < 0.001, respectively), CMP (H(2) = 78.25; p < 0.001, respectively), CMP/R (H(2) = 67.62; p < 0.001, respectively), TMP (H(2) = 77.57; p < 0.001, respectively), and TMP/R (H(2) = 79.12; p < 0.001, respectively) groups. In turn, in the sublingual gland, the above parameter was significantly lower  than that in the submandibular gland for all groups  were significantly higher in all experimental groups (H(4) = 55.70; p < 0.001 and p = 0.002, respectively) than in the control group. In the submandibular gland, the percentage of collagen (n = 30 in each group) was significantly higher only in the CMP and TMP groups (H(4) = 30.26; p < 0.001 and p = 0.018, respectively) vs. control. In turn, in the sublingual gland, the percentage of collagen (n = 30 in each group) was significantly higher in the CMP and CMP/R (H(4) = 73.64; p < 0.001, respectively) groups vs. control. Moreover, in the parotid and submandibular glands in the CMP/R group, the percentage of collagen was significantly lower than that in the CMP group , while in the sublingual gland, no significant differences were noted. In all salivary glands in the TMP/R group, the above parameters were statistically nonsignificant vs. the TMP group. In addition, when comparing the percentage of collagen between the CMP and TMP groups, a lower percentage of collagen fibers was observed in the TMP group than in the CMP group, but the difference was statistically insignificant in all salivary glands  = 57.23; p < 0.001), CMP/R (H(2) = 54.65, p = 0.001), and TMP/R (H(2) = 45.01; p < 0.001) groups and was higher than that in the sublingual gland for the control (H(2) = 57.23; p < 0.001), CMP (H(2) = 50.89; p < 0.001), CMP/R (H(2) = 54.65; p < 0.001), TMP (H(2) = 37.92; p < 0.001), and TMP/R (H(2) = 45.01; p < 0.001) groups. In the sublingual gland, the percentage of these cells was significantly lower  than that in the submandibular gland for all groups  in the parotid, submandibular and sublingual glands were characterized by brown-stained nuclei of acini and ducts  in the CMP and TMP groups was significantly higher in the parotid (H(4) = 44.19; p < 0.001, respectively), submandibular (H(4) = 53.70; p < 0.001, respectively) and sublingual (H(4) = 57.52; p = 0.002 and p < 0.001, respectively) glands than that in the control group. In all studied glands in the CMP/R and TMP/R groups, the percentage of TUNEL-positive cells was significantly lower than that in the CMP  and TMP (p < 0.001) groups  in the CMP and TMP groups was significantly higher in the parotid (H(4) = 27.01; p = 0.006 and p = 0.002, respectively), submandibular (H(4) = 36.15; p < 0.001 and p = 0.001, respectively) and sublingual (H(4) = 48.02; p < 0.001, respectively) glands than that in the control group. In the submandibular and sublingual glands in the CMP/R and TMP/R groups, the percentage of Ki67-positive cells was significantly lower than that in the CMP  and TMP  groups. In the parotid gland, this parameter was significantly lower (p = 0.002) only in the TMP/R group vs. the TMP group  = 31.97; p < 0.001) and CMP/R (H(2) = 47.40, p < 0.001) groups and was lower than that in the sublingual gland for the control (H(2) = 29.27; p < 0.001), TMP (H(2) = 40.88; p < 0.001), and TMP/R (H(2) = 41.35; p < 0.001) groups. In the sublingual gland, the percentage of these cells was significantly higher  than that in the submandibular gland for all groups  in the parotid gland was significantly higher in the CMP, CMP/R and TMP groups (H(4) = 98.55; p < 0.001, respectively) than in the control group. In the submandibular gland, the percentage of PCNA-positive cells (n = 30 in each group) was significantly higher only in the CMP and TMP groups (H(4) = 51.51; p < 0.001, respectively) vs. control. In turn, in the sublingual gland, the percentage of these cells (n = 30 in each group) was significantly higher in all experimental groups (H(4) = 89.79; p < 0.001, respectively) groups vs. control.The percentage of PCNA-positive cells  and TMP  groups  than the CsA-based group. Moreover, in the salivary glands of all experimental groups, focal infiltration of inflammatory cells was observed. It is suggested that fibrosis could be the probable effect of chronic infiltration of inflammatory cells, which is in agreement with data reported by Wynn (2008) .+, Ca2+ and total proteins and also lower activities of antioxidant enzymes after the 60-day treatment with any of the immunosuppressants in the saliva samples collected from parotid and submandibular salivary gland in comparison with the control group. The authors suggest that the prolonged administration of CNIs causes cytological alterations in the salivary glands, which may be related to functional changes. Dehpour et al. (1996) revealed that a 45-day administration of CsA causes both morphological and functional alterations in rat submandibular glands [2+ and K+ concentrations were significantly elevated and pilocarpine-stimulated flow rates were reduced to 54% of those in controls. As a consequence, cytological disorders may be related to abnormal concentrations of selected microelements in the tissue. It is worth emphasizing that the authors did not examine fibrosis in the salivary glands. Our research suggests that FK-506-based regimens appear to have less fibrogenic potential than CsA-based regimens. Research performed by Jain et al. (2000) seems to confirm our observations [The results obtained by other authors seem to confirm our observations, despite the use of CNIs in monotherapy , 17. Spor glands . They obrvations . They reIn our experiment in rats that converted from CNI-based three-drug regimens to rapamycin monotherapy, similar morphological alterations were observed in all salivary glands compared to the animals that were administered CNIs in the three-drug combinations throughout the duration of the experiment. Moreover after the conversion of CsA-based triple therapy to rapamycin, less collagen accumulation was found. In submandibular and sublingual salivary glands, in the FK506-based group converted to rapamycin, tendency to less collagen deposition was also noted compared to scheme without conversion .Our results are in agreement with the findings of Soliman et al. . The autOther authors , 18 haveIn our experiment, in rats treated with CNI-based triple regimens, higher percentages of TUNEL-, Ki67- and PCNA-positive cells were found in the parotid, submandibular and sublingual salivary glands compared to the percentages in the control group. Unfortunately, other authors did not examine the percentage of apoptotic and proliferative cells in salivary glands after immunosuppressive therapy. Only Dehpour et al. , using tIn our experiment in rats that converted from CNI-based three-drug regimens to rapamycin monotherapy, decreased apoptosis and proliferation in all salivary glands were observed compared to the groups without conversion. The antiapoptotic and antiproliferative effects of rapamycin were also noted in our previous studies on the rat prostate , 25 and The available literature contains data on the analysis of the concentration of immunosuppressive drugs in the saliva of patients. The authors indicate that the measurement of the concentration of immunosuppressive drugs in oral fluids seems to be a feasible and non-invasive method for predicting the concentration of drugs in whole blood , 30. TheDuring the experiment, saliva samples were not collected from the animals. Data concerning protein and electrolyte contents in the saliva would undoubtedly complete the presented results.Limitations in the use of the rat model should also be taken into account. The results obtained in rodents cannot be directly translated into humans due to interspecies differences, the varied clinical status of patients, and the medications taken by patients. However, despite these limitations, with proper consideration of the interspecies differences when interpreting the results, the obtained findings may have important clinical implications in patients undergoing immunosuppressive therapy.Long-term administration of immunosuppressive drugs, both in triple regimens based on CNIs and after conversion to rapamycin monotherapy, causes morphological changes in the salivary glands of rats. Consequently, this may have an effect on salivary glands function.Immunosuppressive treatment based on CNIs is associated with an increase in collagen accumulation in all salivary glands. However, FK-506-based regimens appear to have less fibrogenic potential than CsA-based regimens, which may be a key element in the selection of a long-term regimen in patients suffering from various diseases.CNI-based regimens may exert proapoptotic and proliferative effects, while the conversion of treatment from CNI-based regimens to rapamycin monotherapy has antifibrotic, antiapoptotic, and antiproliferative effects on rat salivary glands. These changes may possibly prevent selected abnormalities resulting from the application of CNIs.Supplementary file1 Supplementary Figure 1. Representative light micrographs of hematoxylin and eosin-stained parotid (PRG), submandibular (SMG) and sublingual glands (SLG) of rats in the control, CMP, CMP/R, TMP and TMP/R groups. C − control group without any medication; CMP − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone; CMP/R − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months; PRG – parotid gland; SLG – sublingual gland; SMG – submandibular gland; TMP − rats treated with tacrolimus, mycophenolate mofetil, and prednisone; TMP/R – rats treated with tacrolimus, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months. (TIF 73177 KB)Supplementary file2 Supplementary Figure 2. Representative light micrographs of Masson trichome-stained (blue-stained collagen fibers) parotid (PRG), submandibular (SMG) and sublingual glands (SLG) of rats in the control, CMP, CMP/R, TMP and TMP/R groups. C − control group without any medication; CMP − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone; CMP/R − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months; PRG – parotid gland; SLG – sublingual gland; SMG – submandibular gland; TMP − rats treated with tacrolimus, mycophenolate mofetil, and prednisone; TMP/R – rats treated with tacrolimus, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months. (TIF 73177 KB)Supplementary file3 Supplementary Figure 3. Representative light micrographs of the histochemical reaction showing TUNEL-positive cells in the parotid (PRG), submandibular (SMG) and sublingual glands (SLG) of rats in the control, CMP, CMP/R, TMP and TMP/R groups. C − control group without any medication; CMP − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone; CMP/R − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months; PRG – parotid gland; SLG – sublingual gland; SMG – submandibular gland; TMP − rats treated with tacrolimus, mycophenolate mofetil, and prednisone; TMP/R – rats treated with tacrolimus, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months. (TIF 73227 KB)Supplementary file4 Supplementary Figure 4. Representative light micrographs of the immunohistochemical reaction showing Ki67-positive cells in the parotid (PRG), submandibular (SMG) and sublingual glands (SLG) of rats in the control, CMP, CMP/R, TMP and TMP/R groups. C − control group without any medication; CMP − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone; CMP/R − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months; PRG – parotid gland; SLG – sublingual gland; SMG – submandibular gland; TMP − rats treated with tacrolimus, mycophenolate mofetil, and prednisone; TMP/R – rats treated with tacrolimus, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months. (TIF 73177 KB)Supplementary file5 Supplementary Figure 5. Representative light micrographs of the immunohistochemical reaction showing PCNA-positive cells in the parotid (PRG), submandibular (SMG) and sublingual glands (SLG) of rats in the control, CMP, CMP/R, TMP and TMP/R groups. C − control group without any medication; CMP − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone; CMP/R − rats treated with cyclosporin A, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months; PRG – parotid gland; SLG – sublingual gland; SMG – submandibular gland; TMP − rats treated with tacrolimus, mycophenolate mofetil, and prednisone; TMP/R – rats treated with tacrolimus, mycophenolate mofetil, and prednisone in the first three months of the experiment and rapamycin in the last three months. (TIF 73177 KB)Below is the link to the electronic supplementary material.


Diagnosis of liver disease at earlier stages can improve outcomes and reduce the risk of progression to malignancy. Liver biopsy is the gold standard for diagnosis of liver disease, but is invasive and sample acquisition errors are common. Serum biomarkers for liver function and fibrosis, combined with patient factors, may allow for noninvasive detection of liver disease. In this pilot study, we tested and validated the performance of an algorithm that combines GP73 and LG2m serum biomarkers with age and sex (GLAS) to differentiate between patients with liver disease and healthy individuals in two independent cohorts.n = 260) and healthy subjects (n = 133). The performance of five predictive models using combinations of age, sex, GP73, and/or LG2m from the development cohort were tested. Residual samples from a separate cohort with liver disease  and healthy subjects (n = 106) were used to validate the best performing model.To develop the algorithm, prototype immunoassays were used to measure GP73 and LG2m in residual serum samples collected between 2003 and 2016 from patients with staged fibrosis and cirrhosis of viral or non-viral etiology , which had an AUC of 0.92 (95% CI: 0.90–0.95), a sensitivity of 88.8%, and a specificity of 75.9%. In the validation cohort, the GLAS algorithm had an estimated an AUC of 0.93 (95% CI: 0.90–0.95), a sensitivity of 91.1%, and a specificity of 80.2%. In both cohorts, the GLAS algorithm had high predictive probability for distinguishing between patients with liver disease versus healthy controls.GP73 and LG2m serum biomarkers, when combined with age and sex , showed high sensitivity and specificity for detection of liver disease in two independent cohorts. The GLAS algorithm will need to be validated and refined in larger cohorts and tested in longitudinal studies for differentiating between stable versus advancing liver disease over time.The online version contains supplementary material available at 10.1186/s12014-023-09444-7. Liver disease is a major cause of death in many countries , and patCurrent clinical practice guidelines include liver biopsy as the gold standard for the diagnosis of liver fibrosis and cirrhosis ; howeverGP73 was initially described as a novel Golgi-localized protein that is upregulated in viral and nonviral liver disease . SeveralLaminin-332 Ln-322) is abundant in HCC tissue, where it has been reported to support proliferation, migration, and invasion of tumor cells –19. More is abundIn previous work, we developed and validated a new algorithm for the early detection of HCC that included age, sex, alpha fetoprotein (AFP), and PIVKA-II (ASAP) . Other sMice were immunized with recombinant GP73 and fusions were performed with an NSO myeloma cell line to produce monoclonal antibodies. Twelve IgG antibodies were produced in-house and were screened for use in a prototype GP73 ARCHITECT immunoassay, for a total of approximately 144 antibody pairs. The best antibody pairs were initially selected based on sensitivity, range, and reagent stability. The capture IgG1 (kappa) antibody was coated on magnetic microparticles. The conjugate antibody was murine human chimeric Fab (muhuFab) produced in CHO cells. This conjugate design minimized interference by human anti-mouse antibodies (HAMA) because it lacks the Fc region of the antibody and provided better sensitivity . MicropaA hybridoma of an anti-LG2m monoclonal antibody (Clone 1) used for capture was originally developed by Koshikawa et al. . A hybrin = 246) were purchased from BioIVT , Biomex GmbH , Discovery Life Sciences , and ProMedDx . These three sample sets (JHU/UTSMC/WVC) were combined to develop and train the liver fibrosis and cirrhosis diagnostic algorithm (development cohort). The study was approved by the JHU IRB (#IRB00196747).The prototype GP73 and LG2m ARCHITECT immunoassays were used to measure GP73 and LG2m concentrations in residual serum samples collected between 2003 and 2016 at JHU in Baltimore, MD, from patients with fibrosis or cirrhosis with viral or non-viral etiology, and healthy controls (n = 147) . All fibn = 501). This cohort was used to validate the model derived from the development cohort. Patients with chronic liver disease included those with fatty liver disease, HBV-induced liver disease, and/or autoimmune hepatitis. For each serum sample, the following de-identified data was collected: age, sex, race/ethnicity, and etiology of liver disease. The study was approved by the PUMCH IRB (HS-2386).For the validation cohort, samples were obtained from PUMCH , from patients with liver disease  and healthy subjects . Each two-step sandwich immunoassay utilizes paramagnetic microparticles coated with either anti-GP73  45–63), 56 (IQR 48–63), and 40 (IQR 33–56) years, respectively, with the majority of patients being White males (fibrosis/cirrhosis) or Black males .The development cohort consisted of serum samples from 78 patients with fibrosis (with or without hepatitis), 182 patients with cirrhosis (with or without hepatitis), and 133 healthy subjects  than in healthy controls . On average, patients with cirrhosis had higher median biomarker concentrations  compared to patients with fibrosis GP73 72.12 ng/mL; LG2m 10.46 pg/mL; Fig. p < 0.0001 and p = 0.0003, respectively).Five models were created and compared from the development cohort data using four potential variables: age, sex, GP73, and/or LG2m Table . The AUCp-values < 0.0001). The increase was not statistically different compared to Model 3 with GP73, age, and sex (p = 0.0621).The best model included all four variables, GP73, LG2m, age, and sex , and increased the AUC to 0.92 (Model 5: 95% CI: 0.90–0.95), with a sensitivity of 88.8% and a specificity of 75.9%  was evaluated using the validation cohort data as an independent assessment of clinical performance Table . The GLAThe performance of the GLAS algorithm was assessed by disease state in both the development and validation cohorts Fig. A, B. In In this pilot study, we demonstrated that serum biomarkers GP73 and LG2m, when combined with age and sex to create the GLAS algorithm, showed superior sensitivity and specificity for detection of liver fibrosis and cirrhosis. Analysis of the GLAS algorithm in an independent validation cohort showed similar clinical performance, although with lower AUC, sensitivities, and specificities. Differences in the demographics and disease classifications and etiologies between the development and validation cohorts may account for differences in biomarker levels and model performance; for example, the development cohort had a smaller number of patients with cirrhosis and a more diverse patient population than the validation cohort. Nevertheless, the GLAS algorithm remained robust for distinguishing between healthy subjects and patients with fibrosis or cirrhosis in these two highly different cohorts.The new GLAS algorithm outperformed other algorithms used for diagnosing liver disease that are reported in the literature. In our study, Model 5 had an AUC of 0.92, sensitivity of 88.8%, and specificity of 75.9% in the development cohort and an AUC 0.93, sensitivity of 91.1%, and specificity of 80.2% in the validation cohort for detection of fibrosis or cirrhosis. These AUC values are much higher than those for the FIB-4 (AUC 0.751) and APRI algorithms AUC 0.737) for significant fibrosis   and two demographic variables (age and sex) that demonstrated promise for predicting liver disease (fibrosis and cirrhosis). The GLAS algorithm will be further tested in cohorts with staged fibrosis and cirrhosis to evaluate its performance in early detection of liver disease. The GLAS algorithm will also be compared in head-to-head studies with other leading blood-based algorithms such as FIB4 or APRI, as well as in larger cohorts with greater geographic and racial/ethnic diversity and various liver disease etiologies.Compared to individual biomarkers, the combination of GP73 and LG2m with age and sex significantly improved the accuracy of detecting fibrosis and cirrhosis liver disease in two large and diverse patient cohorts. Further refinement of the GLAS algorithm will produce a highly accurate clinical tool to aid in the evaluation of signs of disease progression in patients with advanced liver disease. Larger longitudinal studies are needed to validate the GLAS algorithm in detecting stable versus advancing liver disease in patients over time.Below is the link to the electronic supplementary material.Additional File 1. Current Biomarker Tests and Algorithms for Liver Disease. Description: Table listing the current biomarkers and algorithms and their performance for diagnosis of liver disease.Additional File 2. GP73 muhuFab/CHO Conjugate Stability: 30 °C X 14 Day & 37 °C X 14 Day comparing IR (incorporation ratio of acridinium per conjugate antibody) range of conjugates. Description: Table showing conjugate stability under various temperature and time conditions.Additional File 3. Microparticle Stability: 45 °C X 3 Days. Description: Table showing microparticle stability under various temperature and time conditions.


This study aims to preliminarily evaluate the effect and safety of electroacupuncture (EA) in treating pediatric chronic urinary retention (CUR) following lumbosacral surgeries, with treatment duration evaluated.This prospective case-series study was performed from August 5, 2017, to July 31, 2022. Pediatric patients diagnosed with CUR following lumbosacral surgeries were included and treated by EA for 2–16 weeks. Responders were defined as participants achieving a reduction of 50% or more in post void residuals (PVR) from baseline. Time-to-event analysis was applied to explore the association between EA treatment duration and response rate. Adverse event was recorded.Totally 14 participants  completed EA treatment. Response rate was 71% (10/14) at the 12th week. 50% (7/14) of participants removed catheters at the 12th week, and none of them experienced re-catheterization in the 24-week follow-up. No serious adverse event was reported. Time-to-event analysis estimated that over 50% patients could respond to EA of more than 8 weeks. Subgroup analysis showed that participants with baseline PVR ≥300 ml and CUR duration ≥12 months experienced longer EA duration to reach the response rate of 50%, compared with those whose PVR <300 ml and CUR duration <12 months .EA could reduce PVR for pediatric patients suffering from CUR following lumbosacral surgeries, with long-term efficacy and safety. EA treatment of more than 8 weeks was reasonable. Further study of a larger sample and controlling is needed.www.chictr.org.cn, identifier, ChiCTR1800020222. Chronic urinary retention (CUR) caused by lower motor neuron lesions (LMNL) is a common but lingering condition among pediatric patients after the surgeries of lumbosacral diseases , 2. For Clinically, CUR means chronically incomplete bladder emptying and large post void residuals (PVR), leading to recurrent urinary tract infections (UTI), stone formation, hydronephrosis and eventual renal deterioration in the long term . LUTD inFirst-line therapy for pediatric CUR includes clean intermittent catheterization (CIC), which is performed several times daily to empty the bladder , 12. HowElectroacupuncture (EA) refers to the practice of inserting multiple fine needles with the addition of electrical stimulation at acupoints, which have specific effects on symptoms, according to Traditional Chinese Medicine (TCM) theory . The effwww.chictr.org.cn (Identifier: ChiCTR1800020222).This was a single-centre and prospective case-series study performed at Guang’anmen Hospital, China Academy of Chinese Medical Sciences from August 5, 2017, to July 31, 2022. This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Guang’anmen Hospital. All participants and their parents or guardians had signed the written informed consent prior to participation. Trial Registration was available at The inclusion criteria were: (1) being younger than 18 years old, (2) being diagnosed with CUR caused by LMNL  following lumbosacral surgeries, (3) being unable to void and dependent on catheterization including CIC or indwelling catheterization (IC), (4) CUR had lasted for 3 months or longer.The exclusion criteria were: (1) bladder outlet obstruction uneliminated, (2) urinary system tumors or stones not removed, (3) major psychological disorders, (4) previous implantation with a cardiac pacemaker or SNM, (5) EA treatment duration of less than 1 week.PVR was measured by urethral catheter output after children’ spontaneous urination attempts without catheterization or assisted bladder emptying . For children with CIC, PVR was the average value of 2 measurements. The interval between two measurements should range from 6 h to 3 days.Bilateral Ciliao (BL32), Zhongliao (BL33), Huiyang (BL35), Shenshu (BL23), and Sanyinjiao (SP6) were inserted with needles. The parameter of needles used at BL32, BL33 and BL35 were 0.30 mm in diameter and 75 mm in length; the needles at BL23 and SP6 were 0.30 mm in diameter and 40 mm in length . Bilateral BL32 and BL33 were needled to a depth of 50 mm–70 mm with an angle of 60°–75° inward and downward, into the second and third sacral foramen. Bilateral BL35 were needled to a depth of 50 mm–60 mm with a slightly superolateral direction. Bilateral BL23 and SP6 were needled vertically to a depth of 25 mm–30 mm. The electric stimulators  were transversely connected to bilateral BL32, BL33, and BL35 with a continuous 5-Hz wave (5–10 mA intensity), while connected to bilateral SP6 with a 10 Hz wave (1–2 mA intensity), with an adjustable current intensity to adapt participants’ tolerance. Needles were retained for 30 min during each treatment session. The participants were treated with EA three sessions a week for consecutive 2–16 weeks. In general, PVR less than 5 ml in children was considered normal and not associated with UTI  were appropriately managed, monitored, and documented by investigators, and were categorized by the acupuncturist and urologists as either acupuncture related or not acupuncture related within 24 h of occurrence. Serious AEs were immediately reported to the principal investigator (L.Z.S.) and the institutional review board at the clinical sites within 24 h.P ≥ 0.05 was thought normally distributed. Paired t-test or paired Fisher exact test were used to compare pre- and post-treatment data for quantitative and binary variables, respectively. Comparison of binary outcomes between subgroups was performed by independent-samples t-test or Fisher exact test. Time-to-event outcome was summarized by Kaplan–Meier estimates. Comparison of time-to-event outcome between subgroups was performed by Log-rank test. Two-sided P value less than 5% was considered significant. Statistical analyses were conducted using STATA software version 14.0  for Windows.Skewed quantitative variables are expressed as median (IQR), and normally distributed variables as mean (SD). The distributions of quantitative variables were assessed for normality by use of the skewness and kurtosis test, and n = 1), neuritis (n = 2), myelitis (n = 2), treatment duration less than 1 week (n = 2)  . A totalAfter EA treatment, the response rate was 71% (10/14) at the 12th week . Around P < 0.001). Likewise, the proportion of participants with severe difficulty in urination was 21% (3/14) with an average of 71% reduction from baseline (P < 0.001). Regarding the proportion of participants with stool retention, it just reduced by 14% from baseline (P = 0.625). After EA treatment, PGI-I score of 1 or 2 corresponding to much better or moderately better was seen in 9 participants (64%). The proportion of participants with catheter removal was 50% (7/14) at the 12th weeks, and none of these participants experienced re-catheterization in the 24-week follow-up. None of the 14 participants had evidence of recurrent UTI, newly emerging hydroureter or hydronephrosis.As displayed in P value was not significant attested by Log-rank test.No significant differences were detected in subgroups of baseline PVR and CUR duration in terms of the primary outcome . HoweverNo episode of serious AEs was reported. Acupuncture-related AEs were infrequent, mild, and transient, which occurred in 2 participants (14%), including sharp pain in 1 participant and localized pigmentation in 1 participant of all cases. No participants discontinued EA treatment because of AEs.To our best knowledge, this is the first time to evaluate the efficacy and safety of EA in treating CUR caused by LMNL following lumbosacral surgeries among pediatric patients and explore the reasonable treatment duration. We found that EA may be a potential treatment option in reducing PVR and facilitating the recovery of bladder voiding function, with long-term efficacy and safety. The reasonable EA treatment duration should be more than 8 consecutive weeks.Typically, the bladder processes the urine in two phases: the filling phase followed by the micturition phase. In a child with voluntary voiding, the micturition phase is initiated through complex neural pathways involving the sacral cord micturition centers at the conus medullaris, cortical and pontine centers in the brain, with final signaling passing through the sacral cord micturition centers (S2–S4) . This siTherefore, the results of the history research are not comparable with this study considering the heterogeneity of different LUTD syndromes and neurologic lesions.It is inspiring that this study shows that EA was effective in reducing PVR by 50% or more in 71.4% of pediatric patients with CUR caused by LNML following lumbosacral surgeries. Moreover, 50% (7/14) participants in our study became capable of stopping catheterization at the 12th weeks and none of these participants experienced re-catheterization in the 24 weeks of follow-up, suggesting EA may be effective in maintaining satisfactory bladder voiding function in the long-term. The mechanism of EA may be based on EA stimulation at BL32 and BL33, activating S2–3 afferent nerve fibers to promote detrusor smooth muscle contractions , althougP value was not significantly attested by Log-rank test, which may be due to small sample size in the single center trial. A multi-center randomized controlled trial of a lager sample should be designed to testify these results. Nevertheless, it did highlight an important knowledge gap regarding the clinical decision of EA treatment and its treatment duration basing on different baseline characteristics of pediatric patients.In addition, our subgroup analysis showed that the response rate at the 12th week was not significantly different across the subgroups of baseline PVR and CUR duration, indicating EA might have relative efficacy and general applicability regardless of the baseline characteristics of patients. Our time-to-event analysis estimated that over 50% participants could respond to EA treatment after 8 weeks of treatment, which was reasonable and consistent with most previous studies , 44. SubThis was the first clinical study to evaluate the efficacy and safety of EA in treating CUR caused by LMNL, following lumbosacral surgeries in pediatric patients. This study drew a conclusion that EA could reduce PVR and facilitate the recovery of bladder voiding function. But it has several limitations. Firstly, urodynamic examination was not conducted although PVR was regarded as the primary objective indicator of the outcome. Secondly, control group was not set as this study was to observe the effectiveness of EA in advance. Sham EA control should be set in further study to eliminate the possible interferences of placebo effects and natural course of disease. Thirdly, this was a single-center case series study of a small sample size, and a multi-center randomized controlled trial of a lager sample size should be designed in the future.EA could be a potential treatment option for pediatric patients diagnosed with CUR caused by LMNL following lumbosacral surgeries. EA may be effective in reducing PVR and facilitating the recovery of bladder voiding function, with long-term efficacy and safety. The reasonable treatment duration of EA should be more than 8 consecutive weeks. Further study of a larger sample and controlling group is needed.


Bilirubin has both antioxidative and prooxidative properties. The study aimed to explore the relationship between serum bilirubin and hemorrhagic transformation (HT) after intravenous thrombolysis in patients with acute ischemic stroke.The patients receiving intravenous thrombolysis with alteplase were retrospectively analyzed. HT was defined as new intracerebral hemorrhage in follow-up computed tomography images within 24–36 h after thrombolysis. Symptomatic intracranial hemorrhage (sICH) was defined as HT accompanied by worsening neurological function. Multivariate logistic regression and spline regression models were performed to investigate the relationship between serum bilirubin levels and the risk of HT and sICH.p = 0.006), direct bilirubin , and indirect bilirubin  had increased risk of HT. Furthermore, multiple-adjusted spline regression models excluded nonlinear association between serum bilirubin levels and HT (p > 0.05 for nonlinearity). Similar results were present between serum bilirubin and sICH.Among 557 included patients, 71 (12.7%) were diagnosed with HT and 28 (5.0%) developed sICH. Patients with HT had significant higher baseline serum total bilirubin, direct bilirubin, and indirect bilirubin levels than those without HT. Multivariable logistic regression analysis indicated that patients with higher serum bilirubin levels, including total bilirubin (OR 1.05, 95% CI 1.01–1.08, The data showed the positively linearly relationship between serum bilirubin levels and the risk of HT and sICH in patients with acute ischemic stroke undergoing intravenous thrombolysis. Intravenous thrombolysis with alteplase is the recommended therapy for acute ischemic stroke (AIS) increasing disability-free survival of approximately 5–10% for patients . HemorrhBilirubin, including direct bilirubin and indirect bilirubin, is the end product of heme catabolism and a potent endogenous antioxidant . A meta-At present, the research on bilirubin and HT after intravenous thrombolysis is scarce. Therefore, we conducted this study to investigate the association of serum bilirubin with HT and sICH after intravenous thrombolysis in patients with AIS. Given the dual antioxidant and prooxidant roles of bilirubin, we further explored whether nonlinear relationship existed between serum bilirubin and HT or sICH.This was a single-center, retrospective and cohort study. The patients with AIS admitted to the Second Affiliated Hospital of Wenzhou Medical University between January 2017 and January 2022 were examined. We enrolled patients who received intravenous thrombolysis using alteplase  within 4.5 h after stroke onset. The diagnosis of AIS was confirmed by brain magnetic resonance imaging (MRI) or computed tomography (CT). Patients who had (1) absence of pre-thrombolytic serum bilirubin levels, (2) absence of follow-up CT within 36 h, (3) thrombolysis with low-dose alteplase or interruption of thrombolysis for reasons other than HT, and (4) hepatitis, cirrhosis, liver cancer, biliary obstruction, renal insufficiency, and hemolysis were excluded. The Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University approved the study. Informed consent was waived for the retrospective nature of the study.The demographic data (age and sex), medical history  and clinical information  were collected. Blood samples were collected from the peripheral vein of each patient before the use of alteplase, including blood glucose, platelet, international normalized ratio (INR), creatinine, hemoglobin A1c (HbA1c), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), albumin, uric acid, total bilirubin, direct bilirubin, and indirect bilirubin. The stroke etiology was determined according to the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) classification. Serum bilirubin levels were measured by a solid-phase chemiluminescent immunometric assay on the SEIMENS ADVIA2400 automatic biochemical analyzer .Patients were followed up for HT and sICH within 36 h after intravenous thrombolysis. HT was defined as a new hemorrhage occurred on the follow-up CT that was not detected by initial CT before intravenous thrombolysis. According to the National Institute of Neurological Disorders and Stroke (NINDS) study, sICH was defined as HT accompanied by deterioration of neurological function . HT can t-test, Mann–Whitney U-test or Kruskal-Wallis test, where appropriate. Categorical variables were presented as absolute values (percentages). The intergroup differences in categorical variables were analyzed with Pearson’s chi-square test or Fisher’s exact probabilities test, where appropriate.The normality of distributions for continuous variables was assessed with Q-Q plot and Kolmogorov–Smirnov test. Continuous variables were presented as mean ± standard deviations (SD) in the case of normally distributed data, or medians (interquartile range [IQR]) in the case of skewed data. The intergroup differences in continuous variables were analyzed with unpaired p < 0.1 in the univariate analysis were included in the multivariate logistic regression model. We included stroke etiology and excluded atrial fibrillation in the model because atrial fibrillation constituted the majority of cardioembolism. Serum bilirubin levels were collapsed into quartiles, and the first quartile values were used as the reference category for the logistic regression analysis. Different serum bilirubin subtypes, including total bilirubin, direct bilirubin and indirect bilirubin, were entered into the models, respectively. Spline regression models were constructed to test whether there was a nonlinear relationship between serum bilirubin and HT or sICH. We set four knots  in the models and the reference point was the median of the first quartile of serum bilirubin. The SPSS 25.0  and R 4.2.1  were used for statistical analyses. A two-sided p value <0.05 was considered statistically significant.Logistic regression model was used to explore the association of serum bilirubin with HT and sICH. Age, sex and other variables with A total of 557 patients with AIS treated with intravenous thrombolysis were finally enrolled and average age was 70 (61–80) years, including 352 (66.5%) men. The average concentrations of serum total bilirubin, direct bilirubin and indirect bilirubin were 16.2 (11.8–20.5), 3.8 (2.7–5.2) and 12.2 (8.8–15.5) μmol/L, respectively. The mean OTT of the patients was 159.6 ± 56.9 min and there were 47 patients (8.9%) with endovascular treatment p = 0.003), direct bilirubin (p < 0.001) and indirect bilirubin (p = 0.015) in patients with HT were significantly higher than those without HT. Similarly, patients with sICH had significantly higher levels of total bilirubin (p = 0.013), direct bilirubin (p = 0.001) and indirect bilirubin (p = 0.026) than patients without sICH. Compared to patients without HT, those with HI had significantly higher levels of direct bilirubin (p = 0.017) and those with PH had significantly higher levels of total bilirubin (p = 0.008), direct bilirubin (p = 0.001) and indirect bilirubin (p = 0.020) (The baseline characteristics of patients stratified by the presence of HT and sICH were shown in = 0.020) . In addip for trend = 0.004). The fully adjusted ORs from the second to the fourth quartile of direct bilirubin were 1.34 (0.51–3.50), 2.79 (1.16–6.72), and 4.18 (1.74–10.00), respectively (p for trend <0.001), compared with the first quartile. As for indirect bilirubin, the fully adjusted ORs from the second to the fourth quartile were 1.54 (0.70–3.37), 1.13 (0.49–2.56), and 2.37 (1.12–5.03), respectively (p for trend = 0.047), compared with the first quartile. The risk of sICH only increased in the fourth quartile of total bilirubin (OR 4.99 95% CI 1.32–18.94), direct bilirubin  and indirect bilirubin  compared with the first quartile of counterparts. Restricted cubic spline regression model excluded the nonlinear association of serum bilirubin with HT and sICH  to high levels of indirect bilirubin increased number of caveolae and levels of caveolin-1 and vascular endothelial growth factor, while prolonged exposure (72 h) disruption led to down-regulation of zonula occludens-1 and β-catenin and thereby damage the tight junctions and cell-to-cell contacts  disruption is a pivotal process in HT resulting in blood extravasation through fragile vessels . Generatcontacts . Oxidaticontacts . Thus, bAs a physiological antioxidant, serum bilirubin levels were increased at acute stage of ischemic stroke in response to oxidative stress. The extent of elevated serum bilirubin levels is positively related to the severity of stroke . Taking The significant difference in serum bilirubin levels between HI and non-HT was only observed in direct bilirubin. This implies that direct bilirubin is superior to total bilirubin and indirect bilirubin for predicting HT after intravenous thrombolysis. Direct bilirubin is more soluble in serum than lipophilic indirect bilirubin after conjugation and bound weakly to albumin, thus making direct bilirubin an active form of bilirubin more readily available than indirect bilirubin .There are several limitations to our study. First, this is a retrospective and single-center study with a moderate sample size. The conclusion of our study needs to be verified in larger cohorts. Second, serum bilirubin was measured at admission and serial bilirubin measurements were not obtained. Whether dynamics of serum bilirubin are associated with HT is unknown. Third, different definition of sICH may have impact on the association between bilirubin and sICH. Due to the retrospective nature of this study, we only adopted one definition of sICH. Finally, functional outcomes and cognitive performance were not evaluated in our study. The impact of serum bilirubin on long-term prognosis in patients with AIS needs further investigation.In conclusion, higher serum bilirubin  levels at admission are associated with HT and sICH after intravenous thrombolysis in patients with AIS. Future studies are needed to clarify the pathophysiological mechanism underlying the association of bilirubin with HT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XH and FF conceived and designed the study. All authors acquired the data, which XC and XY analyzed. XY, XX, XH, and FF assisted in data interpretation and XC wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Despite the fact that the role of endoglin on endothelial cells has been extensively described, its expression and biological role on  cancer cells is still debatable. Especially its function on squamous cell carcinoma (SCC) cells is largely unknown. Therefore, we investigated SCC endoglin expression and function in three types of SCCs; head and neck (HNSCC), esophageal (ESCC) and vulvar (VSCC) cancers. Endoglin expression was evaluated in tumor specimens and 14 patient-derived cell lines. Next to being expressed on angiogenic endothelial cells, endoglin is selectively expressed by individual SCC cells in tumor nests. Patient derived HNSCC, ESCC and VSCC cell lines express varying levels of endoglin with high interpatient variation. To assess the function of endoglin in signaling of TGF-β ligands, endoglin was overexpressed or knocked out or the signaling was blocked using TRC105, an endoglin neutralizing antibody. The endoglin ligand BMP-9 induced strong phosphorylation of SMAD1 independent of expression of the type-I receptor ALK1. Interestingly, we observed that endoglin overexpression leads to strongly increased soluble endoglin levels, which in turn decreases BMP-9 signaling. On the functional level, endoglin, both in a ligand dependent and independent manner, did not influence proliferation or migration of the SCC cells. In conclusion, these data show endoglin expression on individual cells in the tumor nests in SCCs and a role for (soluble) endoglin in paracrine signaling, without directly affecting proliferation or migration in an autocrine manner. BesidesDespite intense treatment regimens, these three types of SCCs show a high number of cancer-related deaths. Therefore, new efficient targeted therapies are needed. Endoglin (CD105), is a co-receptor for ligands of the transforming growth factor beta (TGF-β) pathway and endothelial endoglin expression is associated with metastasis and poor oncological outcome in SCC patients . The TGFin vitro.In multiple types of cancer, it has been shown that a high endoglin expression on tumor vessels is correlated with a worse clinical outcome , 21. Con2.2.1.n = 5), ESCC (n = 11), or VSCC (n = 7). Samples were used in an anonymized manner and according to the guidelines of the Medical Ethical Committee of the LUMC, and conducted in accordance to the Declaration of Helsinki and the Code of Conduct for responsible use of Human Tissue and Medical Research as drawn up by the Federation of Dutch Medical Scientific Societies in 2011. This Code permits the further use of coded residual  tissue and data from the diagnostic process for scientific purposes.Formalin fixed paraffin-embedded (FFPE) tissue samples were obtained from the Department of Surgery, Leiden University Medical Center . Samples were tumor tissue obtained after surgery for HNSCC (2.2.2O2) in methanol for 20 min, and rehydrated. Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin  in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature. The next day, the slides were washed and then incubated with a biotinylated polyclonal rabbit anti goat secondary antibody  for 30 min, washed and incubated with Vectastain complex . The color was developed using a 3,3’diaminobenzidine (DAB) + substrate chromogen system , following the manufacturer instructions. Nuclear staining was performed using hematoxylin . Slides were dehydrated and mounted using entellan .Immunohistochemistry was performed as described before . In shor2.3.Imaging mass cytometry with the Hyperion mass cytometry system was performed as described before . In shor2.4.2 and were tested monthly for mycoplasma contamination.For HNSCC, oral squamous carcinoma (OSC-19) cells, a human SCC cell line derived from tongue tumor and FaDu cells  were used . Ten pat2.5.Total RNA was isolated using the NucleoSpin RNA isolation kit  according to the manufacturer instructions. cDNA was synthesized with the RevertAid First strand cDNA synthesis kit  using 0.5–1.0 μg as RNA input. qPCR was performed with SYBR Green Master mix  using the iCycler Thermal Cycler and iQ5 Multicolour RT-PCR Detection System . Target genes were amplified using specific primers, described in 2.6.Squamous cell carcinoma cells were seeded in six-well plates  and upon 70–80% confluency, they were serum-starved for 7 h in serum-free RPMI 1640 medium. After starvation, the cells were stimulated for 1 h with either 50 ng/mL BMP-6 , 1–2 ng/mL BMP-9 , or 5 ng/mL TGF-β1 . For inhibition studies, 40 μg/mL human IgG  or 40 μg/mL TRC105  was added. The IgG control and the TRC105 antibodies were added 30 min before stimulation with BMP-9.After stimulation, the cells were lysed in RIPA buffer  and protein content was determined via DC protein assay according to the manufacturer instructions . Western blot analysis was performed as described before . Membran2.7.The levels of membrane bound endoglin were measured in cell lysates, while the levels of soluble endoglin were measured in the conditioned medium of the cells by ELISA as described before . In shor2.8.BsmBI-digested plentiCRISPRv2-puromycin [Addgene: 98290  were seeded in 96-well plates  in triplicate. After 16 h, medium was replaced with 100 μL medium with 10% FCS or FCS-free medium supplemented with either 1–2 ng/mL BMP-9  or 5 ng/mL TGF-β1 . At indicated time points, 20 μL MTS substrate  was added to each well and absorbance was measured at 490 nm with the Cytation 5 .2.10.Squamous cell carcinoma cells were seeded in 48-well plate  in triplicate for each condition (300.000 cells/well). After a confluent monolayer was formed, a scratch was made with a 200 μL pipette tip. After placing the scratch, plates were washed, to remove floating cells. Following, either medium with 10% FCS was added, or FCS-free medium supplemented with either 1–2 ng/mL BMP-9 (R&D systems), 5 ng/mL TGF-β1 (Peprotech), 40 μg/mL hIgG (Bio × Cell), or 40 μg/mL TRC105 . Images were obtained at different time points, and the open area of the open surface was quantified with ImageJ software .2.11.t tests were used to compare two groups. One-way ANOVA was used to compare multiple groups. All analyses were performed using GraphPad Prism software . Values of p ≤ 0.05 were considered statistically significant.Data are presented as means ± SD. Unpaired 3.3.1.Despite the fact that endoglin was originally identified as a marker for newly formed endothelial cells, more recent works show selective expression on a variety of cells in various (pathologic) conditions. Endoglin expression on epithelial tumor cells, however, has been a subject of debate for some years, with a limited number of published studies. Therefore, we wanted to analyze endoglin expression in three squamous cell carcinoma types: HNSCC, ESCC, and VSCC. Firstly, we analyzed tumor cell specific endoglin expression in these tumors using patient samples. Immunohistochemical analysis was performed on five HNSCCs, 11 ESCCs, and seven VSCCs. Specific expression of endoglin on epithelial tumor cells was evaluated. Immunohistochemical analysis showed high endoglin expression on newly formed blood vessels, as previously reported, together with tumor type dependent staining of stromal cells in all SSCs evaluated. When epithelial endoglin expression was assessed, we found endoglin expression in nine of the 11 (81.8%) ESCCs, five of the 5 (100%) HNSCCs, and seven of the 7 (100%) VSCCs. We observed that individual cells in the tumor islands show high endoglin expression, while the majority of the SCC cells do not . To explTo confirm that the cells in the tumor expressing endoglin are indeed epithelial cancer cells and not macrophages, which were also shown to express endoglin , we perf3.2.Given that in patient material, individual SCC cells are endoglin positive, but the majority is endoglin negative, we wanted to evaluate how this is reflected in SCC cell lines, to further investigate how endoglin can affect the biological behavior of SSC cells. Therefore, endoglin expression was investigated using qPCR, western blot, and ELISA analysis. Endoglin mRNA was expressed at varying levels in 10 ESCC cell lines (obtained from 10 patients). Out of these 10 cell lines, TE01 and TE15 show relatively high endoglin mRNA expression . However3.3.Having established that SCC cells express endoglin at variable levels, we further investigated how endoglin can affect BMP and TGF-β signaling in SCC cells. In endothelial cells, endoglin is a key regulator for signaling of ligands of the TGF-β family, by shifting the pathway toward the SMAD1 pathway via an interaction with the type I receptor ALK1, instead of ALK5. Therefore, the expression of all known ALKs was evaluated on the SCC cell lines via qPCR. The low endoglin expressing ESCC cell line TE10 shows expression of ALK2, −3, −4, −5, and − 6 while ALK1 and − 7 were not detectable . Similar3.4.Next, we evaluated activity of the endoglin pathway and endoglin dependent regulation of the BMP/TGF-β signaling pathway in SCC cells. Therefore, we used two approaches: Endoglin overexpression (OE) in endoglin low ESCC cells and CRISPR/Cas9 mediated endoglin knockout (KO) in high endoglin expressing ESCC cells. Absence of endoglin expression was confirmed via flow cytometry . The secNext, we set out to investigate the role of SCC epithelial endoglin expression on activity of the TGF-β pathway. ESCC cells were stimulated with the endoglin ligands BMP-9 and TGF-β, which can both signal via endoglin, while TGF-β can also directly induce signaling in an endoglin independent manner through TβRII/ALK5 interaction. This leads to downstream phosphorylation of SMAD1 and SMAD2 signaling molecules, respectively. BMP-6 stimulation, which acts independent of endoglin via a BMPRII/ALK3 interaction, was taken along as a control.Low endoglin expressing ESCC cells TE10 and TE11 showed robust SMAD1 phosphorylation upon BMP-6 and BMP-9 stimulation ,B. SurprTo further explore the role of endoglin in BMP-9 induced signaling, we used an endoglin neutralizing antibody that specifically inhibits BMP-9 binding to endoglin . OSC-19 3.5.Given the varying expression of endoglin on SCC cells and the varying degree to which it is required for signaling, together with reported data that endoglin can affect the migratory capacity of endothelial- and tumor cells via interactions with integrins , we furtThe proliferation of low endoglin expressing TE10 and TE11 ESCC cells was investigated under control conditions and after stimulation with TGF-β and BMP-9. Neither one of the ligands affected proliferation ,C. In coin vitro. A summary of the data, describing the relationship between SCC cell line endoglin expression and BMP-9/TGF-β signaling, cell migration, and cell proliferation, can be found in Next, we evaluated if endoglin can affect SCC cell migration. Cells were seeded and allowed to reach confluence. Subsequently, a wound was made and the wound closure was followed in time. Given the fact that experiments with mitomycin C, to block proliferation, did not show reproducible data (data not shown) and was accompanied by high toxicity, the effects of proliferation on wound closure cannot be completely excluded. Neither the stimulation with TGF-β or BMP-9 or the OE of endoglin affected cell migration in TE10 and TE11 ESCC cells –D. Simil4.In vitro high expression of endoglin by SCC cell lines leads to endoglin shedding and subsequently inhibition of BMP-9 induced signaling. Finally, endoglin does not seem to influence in vitro SCC cell proliferation and migration in vitro. However, this does not necessarily mean that endoglin does not affect SCC migration and proliferation in vivo.Although the role of endoglin on endothelial cells has been extensively reported, its potential function in SCC has not been determined to the same extent, with only a few reports, often limited to cutaneous SCC , 47. OurInterestingly, when we analyzed ESCC, HNSCC, and VSCC patient samples we observed that endoglin expression was restricted to individual cells in the tumor nest. Endoglin has previously been suggested as a cancer stem cell (CSC) marker in renal cell carcinoma (RCC) . CSCs arSquamous cell carcinoma cell lines derived from these SCCs showed variable expression of endoglin. In ESCC, the majority (8/10) of cell lines showed low expression, while in VSCC, exclusively cells with spindle morphology cells show high expression. Surprisingly, none of the SCC cell lines which were analyzed express ALK1. On the contrary, BMP-9 still induced pSMAD1 in most cells. It has however been reported that besides ALK1, BMP-9 can also signal via ALK2  and therOur data show that BMP-9 signaling is mostly unaffected by endoglin expression, using endoglin KO cells and the endoglin neutralizing antibody TRC105. TRC105 was first introduced in 1999, where treatment with TRC105/drug conjugates resulted in human breast tumor xenograft remission and inhibition of tumor angiogenesis  and has Surprisingly we observed that OE of endoglin in low endoglin expressing cells inhibits BMP-9 induced SMAD1 phosphorylation. Endoglin can be shedded from the membrane by MMP-14  and MMP-in vitro cell migration or proliferation of ESCC cell lines. This is in contrast with previous findings that endoglin (ENG) was reported to be a tumor-suppressing gene in ESCC. The authors showed that endoglin OE leads to significantly reduced colony formation efficiency, invasion efficiency and tumorigenicity  endoglin in paracrine signaling in the tumor microenvironment of SCCs.The raw data supporting the conclusions of this article will be made available by the authors, upon reasonable request.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SH, SJ, MT, MG, EJ-M, SC, TH, JB, WD, MS, and LH made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, took part in drafting the article or revising it critically for important intellectual content, agreed to submit to the current journal, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.SH was supported by a research grant from the Flanders Agency for Research and Innovation (VLAIO-HBC.2018.2002).SH was employed by InnoSer België NV, outside the submitted work. LH reports sponsored research grants from TRACON Pharmaceuticals, not related to the work in this study. In addition, LH is coinventor on a patent on the combination of TRC105 with PD1 therapy issued to TRACON.The remaining 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.


Mycobacterium bovis, a zoonotic pathogen with economic significance as it leads to reduced milk and meat production, and high costs for control measures. The Bacillus Calmette-Guérin (BCG) vaccine, primarily used to prevent tuberculosis in humans, has also been studied for controlling bTB. While showing effectiveness in preventing M. bovis infection and disease in cattle, the BCG vaccine can induce non-specific effects on the immune system, enhancing responses to infections caused by unrelated pathogens, and also having non-specific effects on lactation. The aim of this study is to describe both the specific and non-specific effects of BCG vaccination in calves from a commercial dairy herd in central Chile. Diagnosis of M. bovis infection was performed through the IFNγ release assay (IGRA) using ESAT6/CFP-10 and Rv3615c antigens. The records of milk production, somatic cell count (SCC), clinical mastitis (CM) and retained placenta (RP) during the first lactation were compared between vaccinated and non-vaccinated animals. The breed (Holstein Friesian [HF] v/s HF × Swedish Red crossbred [HFSR]) and the season (warm v/s cold) were also analyzed as categorical explanatory variables. Results of IGRA showed significant differences between vaccinated and control groups, indicating a vaccine efficacy of 58.5% at 18 months post vaccination in HFSR crossbred animals. Although milk production did not vary, SCC and CM showed differences between groups, associated to the breed and the season, respectively. When analyzing CM and RP as a whole entity of disease, BCG showed protection in all but the cold season variables. Overall, the BCG vaccine induced protective specific and non-specific effects on health parameters, which may be influenced by the breed of animals and the season. These results provide new features of BCG protection, supporting initiatives for its implementation as a complementary tool in bTB control.Bovine tuberculosis (bTB) is a chronic disease mainly caused by  Mycobacterium bovis that affects a range of domestic and wild animals, including cattle, badgers, deer, and possums. Furthermore, bTB pose a public health risk, as it can be transmitted to humans through consumption of unpasteurized dairy products or inhalation of infectious aerosols (Bovine tuberculosis (bTB) is a chronic bacterial disease mainly caused by aerosols . The disaerosols . ControlM. bovis, existing several licensed formulations primarily used to prevent tuberculosis (TB) in humans, particularly in high-risk populations  is a live attenuated vaccine that was developed from an attenuated strain of ulations . The BCGulations . While tnfection , 6.ex vivo stimulation with E. coli LPS .This work was carried out in a dairy farm in a rural area of the Metropolitan Region, Chile, enrolled for the study due to its high bTB prevalence (72%), as recorded within the sanitary web system of Agricultural and Livestock Service of Chile (SAG). Such prevalence was determined through the caudal fold testing with the bovine Purified Protein Derivatives (PPDB) antigen  and the F1 generation of HF × Swedish Red crossbred (HFSR) calves.5 colony forming units, 0,1 mL) (n = 145), and a control non-vaccinated group (n = 135), in which animals received 0,1 mL of vaccine diluent . The distribution in each group was randomized in every fieldwork.Female calves up to 40 days-old were recruited in two groups, including those vaccinated subcutaneously with BCG Russia substrain , and processed during the same day after arrival at the laboratory. The blood was stimulated with ESAT-6/CFP-10 and Rv3615c peptide cocktails (5 μg/each peptide/mL), Pokeweed mitogen (6 μg/mL) (Applied Biosystems®Bovigam®) and PBS as a control solution , according to manufacturer recommendations. The cut-off value for ESAT-6/CFP-10 and Rv3615c peptide cocktails (DIVA antigens) was an optical density at 450 nm difference ≥ 0.1 after subtraction of the PBS control values from either cocktail (ΔOD450).After an incubation period at 37°C for 18 h, plasma supernatants were harvested and IFN-γ was detected using the Bovigam 2GSeveral visits were made to the farm for accessing productive and sanitary records, until 180 days after the first calving of animals, which occurred in the range of 24–30 months of age. The data was collected individually from each cow, including the identification numbers of animals , date of birth, date of death, date of first delivery, breed, accumulated milk yields (L), monthly somatic cell counts (SCC), episodes of mastitis and other reproductive illnesses.® software (Cooprinsem). Somatic cell count analyses were performed with a Fossomatic 90 (Foss Electric) according to ISO 13366-2/IDF148-2:2006, once or twice a month.The raw milk of cows was collected twice daily with a milking machine  and a bulk tank milk , and productive records were obtained with the CLI-WinThe efficacy of the vaccine (EV) was calculated using the formula EV(%) = ([Rn−Rv]/Rn) × 100 where Rn and Rv are the rates of the IGRA positives in the unvaccinated and vaccinated groups, respectively . This, aThe accumulated lactation yields at 180 days were calculated through the following formulae .Where:MY: accumulated milk production.I0: days between the start date of the lactation period and the date of the first record.In: days between the last record date and the end of the lactation period.post hoc Tukey’s test.Differences between groups were analyzed with the ANOVA and The SCC data during first 180 lactation days was transformed to a linear score using the following formula .The scores were analyzed for normality with Shapiro-Wilks test, and differences between groups were determined with the Kruskall-Wallis test. In this analysis, SCC counts of animals with a clinical mastitis episode were not included.Y) is dichotomous, because it can only take two values, where Y = 0 and Y = 1 represent the absence and presence of one or both pathogens in each BPS, respectively. Statistical significance was established at p < 0.05.Pathologies recorded until the 180-lactation day were analyzed, which included clinical mastitis (CM) and retained placenta (RP). Diagnosis of CM was made through a daily macroscopic milk analysis, and RP was determined by a physical examination 24 h after parturition. Events of disease, but not their duration, were recorded for analysis. If a cow had two or more events of mastitis, it was considered only once. Two multivariable logistic regression model were constructed (one for CM and other for RP) to evaluate the association between registered variables with the outcomes . In thes® (version 2020).All the statistical analysis were performed with InfoStat softwaren = 110, 57 BCG and 53 control) and HFSR   .p = 0.013) between vaccinated and control groups in HFSR crossbred animals only, showing a high EV% at 18 months post vaccination  or there were significant inconsistencies in their records (n = 2). These analyses finally included 123 BCG vaccinated and 113 control animals.Some animals were excluded from the analysis of post-partum variables, because they did not give birth (p > 0.05) between BCG vaccinated and control individuals (p = 0.04). When the season and overall data were analyzed, no differences were determined   . When th = 0.04) .Mycobacterium bovis . The CM caused by bacterial infections corresponds to a major economic loss in dairy farms, mainly due to diminished milk production, treatment costs and loss of animals (E. coli and coagulase-negative Staphylococci among CM causing bacteria (p = 0.06) when compared with the control group, in accordance with the SCC result, a contrast that was not observed with HF animals  (Permit N°22555 VET-UCH), Universidad de Chile. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.CC: Formal analysis, Investigation, Methodology, Writing – original draft. RA-M: Formal analysis, Methodology, Writing – review & editing. MD: Methodology, Writing – review & editing. PÁ: Supervision, Writing – review & editing. RL: Data curation, Supervision, Visualization, Writing – review & editing. PR: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Writing – review & editing.