The aim of this study was to examine changes in perceived social support from early pregnancy to 2 years postpartum and to test whether these changes (a) differ between mothers and fathers or (b) vary as a function of the Big Five personality traits.Higher peripartum social support in (expectant) mothers and fathers has been associated with fewer complications during pregnancy and delivery as well as better parental and offspring health.N = 396, including n = 293 mothers and n = 103 fathers) and DREAM . The Big Five personality traits were assessed during pregnancy in MARI as well as 8 weeks after the anticipated birth date in DREAM with short forms of the Big Five Inventory. Perceived social support was assessed during pregnancy, 4 months postpartum, and 16 months postpartum in MARI as well as during pregnancy, 14 months postpartum, and 2 years postpartum in DREAM using the short version of the Social Support Questionnaire.Prospective-longitudinal data from two regional-epidemiological samples from Germany were used: MARI (Multilevel analyses revealed that perceived social support decreased across the peripartum period, and this decrease did not differ between mothers and fathers. More extraverted, emotionally stable, agreeable, conscientious, and open parents perceived higher levels of social support across the peripartum period. The peripartum decrease of perceived social support was smaller in parents who were more extraverted.Our findings suggest that especially extraversion plays an important role for high and stable levels of perceived social support across the peripartum period.Particularly highly introverted parents might profit from targeted social support interventions. Before and after the birth of a child, (expectant) mothers and fathers are faced with far-reaching life changes and new responsibilities –4. SociaTherefore, a major research task is to examine what contributes to high and stable levels of perceived social support across the peripartum period. Such research provides important cues for an early identification of high-risk groups of (expectant) parents as well as early targeted interventions to foster peripartum social support.After the birth of a child, the social network size of new parents tends to decrease , while tIn line with traditional gender stereotypes , men migFurthermore, levels and changes of perceived social support across the peripartum period might vary as a function of parents' personality. Personality refers to individual differences in thoughts, feelings, and behaviors  that canPrevious research suggests that higher extraversion, emotional stability, agreeableness, conscientiousness, and openness relate to higher perceived social support . ExtraveEmotionally stable individuals tend to be resistant against stress and experience less negative affect , 26. TheAgreeable individuals are compliant, kind, and supportive . BecauseConscientious individuals are careful, competent, diligent, and organized. Moreover, higher conscientiousness relates to higher mastery and more effective self-regulation , 32. ThuIndividuals who score high on openness tend to be open-minded, adventurous, and creative , 34. TheTaken together, prior findings suggest that (a) perceived social support tends to decrease from pregnancy to postpartum and that (b) higher extraversion, emotional stability, agreeableness, conscientiousness, and openness relate to higher levels of perceived support. However, whether these personality characteristics relate to more favorable changes in perceived social support across the peripartum period  remains an open question so far.We used data from two prospective-longitudinal studies to examine changes in perceived social support from early pregnancy to 2 years postpartum and to test whether these changes (a) differ between mothers and fathers or (b) vary as a function of the Big Five personality traits. We hypothesized that social support decreases from pregnancy to postpartum and that this decrease is smaller in more extraverted, emotionally stable, agreeable, conscientious, and open parents. Gender differences between mothers and fathers were tested exploratorily. The findings from both studies were compared to evaluate the replicability, robustness, and reproducibility of the effects .Prospective-longitudinal data from two regional-epidemiological samples (MARI and DREAM) were combined.The Maternal Anxiety in Relation to Infant Development (MARI) Study is a prospective-longitudinal regional-epidemiological study in (expectant) mothers and their partners from the area of Dresden, Germany (01/2009–09/2012). The study objective was to investigate the role of anxiety and depressive disorders prior to, during, and after pregnancy for perinatal outcomes, maternal health, and offspring development. Seven assessment waves with standardized diagnostic interviews, questionnaires, and observations were conducted: T1 , T2 (week 22–24 of gestation), T3 (week 35–37 of gestation), T4 (10 days postpartum), T5 (2 months postpartum), T6 (4 months postpartum), and T7 (16 months postpartum). More detailed information on the aims, methods, design, and inclusion/ exclusion criteria of the MARI study, including a detailed study flow chart, has been presented elsewhere .Informed consent was obtained from all participants. The study was approved by the Ethics Committee of the Faculty of Medicine of the Technische Universität Dresden (No: EK 94042007) and carried out in accordance with the Helsinki Declaration of 1975, as revised in 2008 and 2013.N = 8); age < 18 or > 40 years (N = 8); multiple pregnancy (N = 2); history of more than three spontaneous abortions, (induced) termination of the pregnancy, still birth, or infant impairment (N = 2); invasive fertility treatment (N = 9); severe physical disease, microsomia, or skeletal malformation (N = 6); substance abuse or heroin substitution during the past 6 months (N = 0); severe psychiatric illness (N = 2); expectation to leave the area of Dresden (N = 6); insufficient German skills (N = 7). Nine women did not participate due to spontaneous abortions before T1, 10 because their partner did not agree, 154 due to a lack of time, and four due to unknown reasons.Overall, 533 expectant mothers were sampled from 22 gynecological outpatient settings in and around Dresden, Germany, and screened for inclusion and exclusion criteria. Fifty women were not enrolled due to the following exclusion criteria: Gestational age > 12 weeks . The participation of eight women ended after T1 due to a spontaneous abortion or termination of the pregnancy. During the study, three women moved away from the area of Dresden, five women could not be reached anymore by phone, postal, or personal contact, nine women reported a lack of time or interest in further participation, and seven women refused to be contacted again for further follow-up assessments. Some retained women did not participate in single assessments, for example, due to a preterm delivery, sickness, or a lack of time. Detailed information on sociodemographic, gynecological, and clinical characteristics of the sample of mothers has been previously published , 39.From week 22 to 24 of gestation, all 306 expectant mothers were asked whether the study personnel may approach their partner to participate in the study, and 134 (43.8 %) indicated that their partner may be contacted by phone. Of these partners, 109 met the inclusion criteria (sufficient German skills as well as willingness and time to participate) and agreed to participate. All 109 partners were men. In total, 109 (expectant) fathers participated at T2, 96 at T5, 91 at T6, and 26 at T7.N = 109) and without (N = 197) a participating partner differed in terms of sociodemographic, psychosocial, and neonatal characteristics ((Expectant) mothers with  is a prospective-longitudinal cohort study in (expectant) mothers and their partners, recruited from June 2017 to the end of 2020 from the area of Dresden, Germany (06/2017–ongoing). The study objective was to prospectively examine the association between parental work participation, role distribution, and stress across the peripartum period, including their effects on perinatal outcomes and family health. So far, six questionnaire-based assessment waves are being conducted: T1 , T2 (8 weeks after the anticipated birth date), T3 (14 months postpartum), T4 (2 years postpartum), T5 (3 years postpartum), and T6 . More detailed information on the aims, methods, design, and inclusion/ exclusion criteria of the DREAM study, including a detailed study flow chart, has been published elsewhere  and carried out in accordance with the Helsinki Declaration of 1975, as revised in 2008 and 2013. The data were collected and are managed using Research Electronic Data Capture (REDCap), hosted at the “Koordinierungszentrum für Klinische Studien” at the Faculty of Medicine of the Technische Universität Dresden , 42.Expectant mothers and their partners were recruited during pregnancy, predominately at information sessions in hospitals and birth preparation courses in and around Dresden, Germany. Inclusion criteria were a current pregnancy, being a resident in the Dresden area, and sufficient German skills to complete the study questionnaires. Specific sub-groups  were not excluded to obtain a diverse sample and maximize the generalizability to the general population of expectant parents. The data collection of the DREAM study is currently still ongoing except for T1. Thus, the sample sizes from T2 onwards do not reflect the finally retained participants but only refer to those parents who have been due at the time of the data extraction. The current study is based on version 8 of the quality-assured data files, released for research in March 2021. Based on this version, 2,209, 1,967, 1,428, and 797 mothers as well as 1,608, 1,343, 969, and 544 partners participated at T1, T2, T3, and T4, respectively. Detailed information on sociodemographic, gynecological, and clinical sample characteristics has been presented elsewhere .In MARI, the Big Five were assessed at T2 (week 22–24 of gestation) with the German version of the BFI-K, the 21-item short form of the Big Five Inventory . The BFIIn DREAM, the Big Five were assessed at T2 (8 weeks after the anticipated birth date) with the German version of the BFI-S, the 15-item short form of the Big Five Inventory  , 45. ThePerceived social support was assessed in MARI at T2 (in week 22–24 of gestation), T6 (4 months postpartum), and T7 (16 months postpartum) and in DREAM at T1 (during pregnancy), T3 (14 months postpartum), and T4 (2 years postpartum) with the short version (K-14) of the Social Support Questionnaire (F-SozU) Form A . The F-SN = 293 mothers and N = 103 fathers). Specifically, 396 individuals (N = 293 mothers and N = 103 fathers) provided information on perceived social support at T2, 358 (N = 281 mothers and N = 77 fathers) at T6, and 280 (N = 255 mothers and N = 25 fathers) at T7.Only individuals with information on personality (at T2 in MARI and DREAM) and perceived social support at any wave  were considered. In MARI, this resulted in a total sample of 396 individuals . Of these individuals, 335 were excluded because they did not complete T1 during (but after) pregnancy. In addition, 9 partners were excluded because they were female and 7 partners were excluded because they were not the biological father of the child. Finally, 14 individuals were excluded due to missing information on their date of birth. Thus, the final sample in DREAM consisted of 2,819 individuals . Specifically, 2,819 individuals  provided information on perceived social support at T1, 1,829  at T3, and 914 (N = 556 mothers and N = 358 fathers) at T4.In DREAM, this resulted in a sample of 3,172 individuals  the respective Big Five trait as well as interaction terms of the respective Big Five trait with (b) gender, (c) the early-postpartum variable, and (d) the late-postpartum variable. A separate model was built for each Big Five trait to avoid multicollinearity. The interaction term with gender was used to test whether the role of the respective Big Five trait for perceived social support differed between mothers and fathers. The interaction terms with the early- and late-postpartum variable, respectively, were used to test whether the short- and/ or long-term changes in perceived social support varied by the respective Big Five trait. r = 0.75 and r = 0.68, respectively. The correlation of perceived social support 4 months and 16 months postpartum was r = 0.74. Perceived social support during pregnancy was slightly higher in parents who did  vs. did not  participate in the study from pregnancy until 16 months postpartum, t(394) = −2.48, p = 0.013. That is, participants with lower initial levels of perceived social support were more likely to drop out.Baseline sample characteristics for mothers and fathers of the MARI and DREAM sample are shown in r = 0.72 and r = 0.65, respectively. The correlation of perceived social support 14 months and 2 years postpartum was r = 0.72. Perceived social support during pregnancy did not differ significantly between parents who did  and did not  participate until 2 years postpartum, t = −0.56, p >0.05. That is, attrition did not vary by initial levels of perceived social support.In DREAM, the correlation of perceived social support during pregnancy with perceived social support 14 months and 2 years postpartum was p-values > 0.05). That is, neither in MARI nor in DREAM, these changes differed between (expectant) mothers and fathers.Means and standard deviations for perceived social support at different assessment waves in the MARI and DREAM sample are shown in p-values >0.05). That is, neither in MARI nor in DREAM, these changes differed between mothers and fathers.Examining the role of personality revealed positive main effects for each of the Big Five personality traits in both studies : Higher p-value >0.05). Moreover, there was no evidence that any other peripartum changes in perceived social support varied as a function of the Big Five personality traits .In DREAM, there was a significant interactive effect between extraversion and the early-postpartum variable (β = 0.04), indicating that more extraverted parents experienced a smaller decrease of perceived social support from pregnancy to 14 months postpartum. However, no such interactive effect was found in MARI  how perceived social support changes across the peripartum period in (expectant) mothers and fathers and (b) whether these changes vary by personality. Our main findings were that both mothers and fathers experienced a decrease of perceived social support from pregnancy to early and late postpartum. More extraverted, emotionally stable, agreeable, conscientious, and open individuals perceived higher levels of peripartum social support. In parents who scored higher on extraversion, the peripartum decrease of perceived social support was smaller.First, we found that fathers experienced lower levels of perceived social support compared to mothers. As suggested by previous research , men migSecond, our findings revealed that perceived social support decreased from pregnancy to early postpartum and from early to late postpartum. These results are consistent with previous findings , 18, 20 Third, we found that more extraverted, emotionally stable, agreeable, conscientious, and open parents felt socially better supported across the peripartum period. As suggested by previous research, higher levels on these traits relate to a range of cognitive, emotional, and interpersonal skills , 26, 55,Fourth, our findings particularly emphasize the importance of extraversion. Extraversion was not only the strongest predictor of perceived social support but also predicted a smaller peripartum decrease of perceived social support. In this regard, it is plausible to assume that more extraverted individuals not only have a larger social network . They miFinally, our findings were nearly identical for both samples, which underlines the replicability of our results. This fact is particularly noteworthy given the “replication crisis” and increasing claims for more replicable, robust, and reproducible research in psychological science . HoweverOur work has several strengths: We used data from two prospective-longitudinal studies in expectant parents. In both studies, the Big Five personality traits were assessed with the BFI, and perceived social support was assessed repeatedly from early pregnancy to late postpartum with the FSozU, a well-established questionnaire. Our findings were highly similar in both samples, which emphasizes the replicability, robustness, and reproducibility of the results.via participating mothers, which might have doubled selection effects and, for example, might have led to an overrepresentation of particularly engaged fathers  and BFI-S (DREAM)—short questionnaires that are less comprehensive than longer measures and, for example, do not allow to distinguish between different facets of the Big Five. Second, in DREAM, personality (T2) was assessed after the first assessment of perceived social support (T1), and personality might have changed from T1 to T2. However, the Big Five traits are considered to be relatively stable over short periods of time –59. Thir fathers . Fifth,  fathers . Sixth,  fathers , 40. ThuTaken together, our findings suggest that especially extraversion plays an important role for high and stable levels of perceived social support across the peripartum period. Therefore, particularly highly introverted mothers and fathers might profit from targeted interventions. Such programs might promote specific skills to establish and maintain social contacts, communicate individual needs, and actively seek for support in stressful situations . Future julia.martini@tu-dresden.de.The data of this study are not openly accessible because after consulting the Ethics Committee and due to the sensitive nature of the questions asked in this study, participants were assured that all raw data would remain confidential and not be shared. Further information on the data can be obtained from the corresponding author, the Ethics Committee of the Medical Faculty of the Technische Universitaet Dresden, and the Institute of Clinical Psychology and Psychotherapy of the Technische Universitaet Dresden. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by the Ethics Committee of the Medical Faculty of the Technische Universitaet Dresden. All participants provided their written informed consent to participate in this study.JM: conception and design of the MARI study and data collection MARI study. SG-N: conception and design of the DREAM study and data collection DREAM study. EA: data analysis and preparation of the manuscript. EA, SG-N, and JM: interpretation, critical review, and approval of the final manuscript. Principle investigator of the DREAM study is SG-N, who is a management committee member of COST action CA18211: DEVOTION: Perinatal Mental Health and Birth-Related Trauma: Maximizing best practice and optimal outcomes. All authors contributed to the article and approved the submitted version.The MARI study was funded by the Institute of Clinical Psychology and Psychotherapy, Technische Universität Dresden, Germany, and supported in parts by the Lundbeck Institute Skodsborg, Denmark. Parts of the field work were additionally supported by the Friends and Sponsors (Gesellschaft der Freunde und Förderer) of the Technische Universität Dresden, Germany. The DREAM study was funded by the German Research Foundation . This paper contributes to the EU COST Action 18211 supported by COST (European Cooperation in Science and Technology).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.


Hyponatremia is the most common electrolyte disorder, commonly affecting older hospitalized individuals; however, the literature is not clear regarding its effect on mortality. The aim of this 2-year observational prospective cohort study was to evaluate the mortality and re-admission rates, the clinical and laboratory characteristics and the causes of hyponatremia in patients older than 65 years admitted with a corrected serum sodium of 130 mEq/L or less in an internal medicine ward of a tertiary Greek university hospital. During the observation period, 138 patients  fulfilled the inclusion criteria and were prospectively followed for 1 year after admission. Symptoms of hyponatremia were present in 59.4% of patients. Hypovolemia was the main sole cause of hyponatremia, but in about one third of patients, hyponatremia was multifactorial. Only a low proportion of patients (12.3%) fulfilled the criteria of the syndrome of inappropriate antidiuresis (SIAD) at admission according to the current guidelines. The re-admission rates at 3- and 12-months following discharge was 34.2% and 51.8%, respectively. Mortality during hospitalization was 17.4% and was higher compared to non-hyponatremic admitted older patients, while the total mortality at 1 year after admission was 28.3%, indicating that hyponatremia at admission is a marker of significant mortality during and after hospitalization in elderly patients. Hyponatremia is the most common electrolyte disorder in both community and hospitalized patients and is associated with adverse outcomes and mortality, especially when it is moderate or severe  . A commoHyponatremia is the most common electrolyte disorder but its reported frequency varies according to the healthcare setting and the definition of hyponatremia ,8. AdditThis is a 2-year prospective cohort study of patients admitted to the Internal Medicine Department of the tertiary University Hospital of Heraklion, Crete, Greece. The study was approved by the ethics scientific committee of the hospital.Patients admitted to the Internal Medicine Department;Age 65 years or more;Moderate (serum sodium 126 mEq/L–130 mEq/L) or profound hyponatremia . We have chosen the threshold of 130 mEq/L as it is associated with increased adverse outcomes and mortality in the general population and difficulties in correction [Inclusion criteria:Non-hypotonic hyponatremia  ;Serum sodium >130 mEq/L after correction for glucose according to the Hiller equation .Exclusion criteria:The flowchart of patient inclusion is presented in Data regarding demographics , medical history, previous episodes of hyponatremia, clinical and laboratory parameters associated with hyponatremia , cortisol, proteins, cholesterol and triglycerides in serum and sodium, creatinine, urea and uric acid in urine) were collected. Presence of symptoms was assessed at admission and included all symptoms associated with hyponatremia . In the case where symptoms could be either a cause or a consequence of hyponatremia , a decision regarding whether the symptom was due to hyponatremia was made on clinical grounds based on the final diagnosis and the clinical response after resolution of hyponatremia. Functional assessment for activities of daily living (ADLs) was performed at the time of admission using the modified Barthel ADL index .Patients were followed up to 12 months after admission and patient outcomes including mortality and re-admissions within 3 and 12 months were noted. If a patient had more than one admission, only the first admission was included in analyses.2 determined according to the EPI-CKD  diet. After that, the algorithm is based on urine biochemistry, followed by the patient’s volume for further identification of the cause of hyponatremia . A patieequation ) was usep-values < 0.05 were considered to be significant. A univariate linear regression analysis was conducted to identify clinical, epidemiological and laboratory factors associated with mortality of hyponatremic patients during admission. All abovementioned statistics were calculated with GraphPad Prism 6.0 . A multivariate logistic regression analysis was conducted to evaluate the effect of factors that were previously identified in the univariate analysis model as factors with a p-value lower than 0.05. Multivariate analysis was performed using SPSS version 23.0 .Data are presented as number (%) for categorical variables and median  or mean  for continuous variables. Categorical data were analyzed with Fisher’s exact test or the Pearson’s chi-square test, as appropriate. Continuous variables were compared using Student’s t-test for normally distributed variables and the Mann–Whitney U-test for non-normally distributed variables. All tests were two-tailed and n = 1577) of patients older than 65 years old fulfilled the inclusion criteria. Seven of these cases were re-admissions and were excluded from the study, while in nine cases blood or urine chemistry at admission was unavailable, resulting in a final sample of 138 patients with hyponatremia (p < 0.0001), confirming that older patients with hyponatremia were more likely to be female.During the 2-year study period, 154 out of 2833 admissions , nausea (56.1%), vomiting (42.7%) and headache (13.4%). Notably, 10.1% of patients were confused at admission and 9.4% had had a fall during the last 14 days before presentation.Hypovolemia was the main sole cause of hyponatremia (28.3%). Thiazide use was detected as the sole cause of hyponatremia in 9.4% (13 patients) and as a main cause of hyponatremia in 21.1% (38 patients) of the sample. In 31.9% of all patients (44 patients), multifactorial hyponatremia (at least two causes of hyponatremia) was found .p = 0.049). In our cohort, the re-admission rate at 3- and 12-months following discharge was 34.2% and 51.8% . Among the 114 patients who were initially discharged, 28.9% had at least one episode of hyponatremia within the next 12 months. The mortality rate at 3- and 12-months following discharge was 6.1% and 13.2% , whereas the total mortality at the end of the 12-month follow-up period in the whole sample of hyponatremic patients was 28.3% (39 out of 138 patients). Serum sodium levels at admission lower than or equal to 125 mEq/L were not associated with increased length of stay, re-admission or mortality rates compared with sodium levels 126–130 mEq/L (The median length of stay was 6 days (IQR 4–9 days). The mortality rate during hospitalization was 17.4% (24 patients out of 138 patients), which was significantly higher than the mortality rate observed in hospitalized patients without hyponatremia  during the same period of observation  of our cohort of patients with hyponatremia. Their main clinical and biochemical characteristics are shown in Patients with thiazide-associated hyponatremia (as a sole or main cause of hyponatremia) were more likely to have a higher Barthel index and to be symptomatic. Furthermore, they had lower corrected serum sodium and they also had lower mortality, lower re-admission rate at 12 months, lower mortality at 12 months and lower incidence of future hyponatremia.Regression analysis was performed in order to identify factors independently associated with mortality in hyponatremic patients. Initially, a univariate linear regression analysis of age, gender, duration of hospitalization, presence of symptoms of hyponatremia, thiazide use, Barthel Index, diagnosis of SIAD during this hospitalization, laboratory parameters in serum such as corrected sodium, urea, uric acid, proteins, creatinine, TSH, chloride, osmolarity and laboratory parameters in urine such as urea, uric acid and sodium with mortality during admission was carried out. Then, we performed a multivariate logistic regression analysis with factors identified in the univariate analysis  to be associated with mortality. The analysis identified thiazide use and increased Barthel Index to be independently negatively associated with mortality during admission and increased serum uric acid to be independently positively associated with mortality during admission. The results of the regression analysis are shown in To the best of our knowledge, this is the first prospective study specifically aimed at providing data on hyponatremic elderly patients regarding re-admission and mortality rates at 1 year follow-up after admission in a medical ward. Additionally, to the best of our knowledge, this is the first study in Southern Europe describing the characteristics and outcomes of hyponatremia in elderly admitted patients.+ < 130 mEq/L) is associated with a higher likelihood of delirium and an elevated in-hospital mortality  [Mortality during admission is rather variable in previous reports of hospitalized patients with hyponatremia, ranging between 5.9% and 27.0%, possibly due to different sample characteristics ,20,21. I= 0.005) . Interes= 0.005) . The ide= 0.005) ,26,27,28In line with other studies, almost 1 out of 3 patients in our cohort had a previous history of hyponatremia . FurtherIn our cohort, more hyponatremic patients were women ; this is in line with other studies in the general population, even though evidence suggests that the female gender as a risk factor may be confounded by low body weight ,29. Our Symptoms of hyponatremia in our study were similar to those in the literature . An impoIn our study, which included elderly patients with moderate/profound hyponatremia, hypovolemia was the most common sole etiologic factor. This is not the case in younger individuals . Althoug2, that could, theoretically, have been used as a cutoff point instead of 30 mL/min/1.73 m2 that was used, since, according to the KDIGO and KDOQI guidelines, persistence of eGFR below 60 mL/min/1.73 m2 for at least three months, irrespective of the cause, is sufficient to diagnose chronic kidney disease [2, since we believe that it more precisely reflects advanced kidney disease, in which the underlying subclinical tubulointerstitial damage that affects urine-concentrating ability would indeed be present. If an eGFR lower than 60 mL/min/1.73 m2 had been used, the rate of SIAD would have been lower, with only 13 patients (9.4%) diagnosed with SIAD.Diagnosing SIAD may sometimes be difficult . The rat disease ,46. In tThiazide-associated hyponatremia seems to have some specific characteristics that make it quite different from the other causes of hyponatremia in elderly patients. Patients with thiazide-associated hyponatremia are more frequently women, more likely to be symptomatic, have lower serum sodium and have a better clinical course with a lower mortality and a lower recurrence rate. Furthermore, thiazide use was also found with the regression analysis to be independently associated with reduced mortality. To our knowledge, there has been no study until now directly comparing the characteristics of the patients with and without thiazide-associated hyponatremia in geriatric patients, even though there are studies describing the characteristics of patients with thiazide-associated hyponatremia both in the general population and in older individuals ,47,48.2 from the diagnosis of SIAD may seem arbitrary, however, the guidelines published by Spasovski et al. do not define a cutoff for the eGFR. Moreover, patients with mild hyponatremia, or patients with hyponatremia occurring in the hospital, were not included in this study. Finally, we do not have data for morbidity/mortality extending after the 12-month period after admission, thus the possible longer-term consequences of hyponatremia cannot be assessed, while, on the other hand, the causes of mortality were not reported.Our study has some limitations that we should acknowledge. First of all, this study included only patients hospitalized in the Internal Medicine Department, thus, patients not admitted, as well as patients admitted in other wards may have been missed. However, it is rather uncommon for patients with moderate or profound hyponatremia to be discharged and most medical patients with such low sodium values are admitted in our department. Furthermore, a few patients with hyponatremia that did not have urine chemistry available were excluded from the analysis. In addition, despite the fact that our hospital is a referral center for Southern Greece, a few post-discharge visits to other healthcare centers may have been missed and therefore the re-admission rate may have been underestimated. Moreover, assessment of hyponatremia relies on the estimation of fluid volume and this can be problematic, since differentiation of hypovolemia from euvolemia can be difficult. Thus, any mistakes during the estimation of the patients’ fluid status may have affected the results of the current study. Additionally, it should be noted that choosing to exclude patients with an eGFR lower than 30 mL/min/1.73 mTo conclude, a significant proportion of older patients (approximately 25%) with hyponatremia were re-admitted or died during hospitalization or in the following 12 months after admission. Thus, hyponatremia on admission is associated with increased morbidity/mortality during the hospital stay and in the next year in elderly individuals. This study also underlines the difficulty of assessing geriatric patients who exhibit multifactorial hyponatremia in a significant portion.


Inflammatory lung disorders (ILDs) are one of the world’s major reasons for fatalities and sickness, impacting millions of individuals of all ages and constituting a severe and pervasive health hazard. Asthma, lung cancer, bronchiectasis, pulmonary fibrosis acute respiratory distress syndrome, and COPD all include inflammation as a significant component. Microbe invasions, as well as the damage and even death of host cells, can cause and sustain inflammation. To counteract the negative consequences of irritants, the airways are equipped with cellular and host defense immunological systems that block the cellular entrance of these irritants or eliminate them from airway regions by triggering the immune system. Failure to activate the host defense system will trigger chronic inflammatory cataracts, leading to permanent lung damage. This damage makes the lungs more susceptible to various respiratory diseases. There are certain restrictions of the available therapy for lung illnesses. Vitamins are nutritional molecules that are required for optimal health but are not produced by the human body. Cholecalciferol (Vitamin D) is classified as a vitamin, although it is a hormone. Vitamin D is thought to perform a function in bone and calcium homeostasis. Recent research has found that vitamin D can perform a variety of cellular processes, including cellular proliferation; differentiation; wound repair; healing; and regulatory systems, such as the immune response, immunological, and inflammation. The actions of vitamin D on inflammatory cells are dissected in this review, as well as their clinical significance in respiratory illnesses. The majority of the world’s population is affected by pulmonary system inflammation, which is both a serious public health issue and a substantial financial burden. Approximately 510 million individuals are thought to be affected by these diseases globally. Inflammatory lung disorders (ILDs) are one of the world’s major reasons for fatalities and sickness, impacting millions of individuals of all ages and constituting a severe and pervasive health hazard. During the onset and progression of many lung diseases, structural aberrations in several sections of the lung, including the nasal passages and airways, are evident. In both small and large airways, this comprises airway remodeling, parenchymal fibrosis, and epithelial lesions. Additionally, cell proliferation, excessive mucus formation, enhanced collagen accumulation, and the stimulation of molecular pathways such as oxidative stress and inflammation all contribute to the loss of elasticity. Asthma, lung cancer, bronchiectasis, pulmonary fibrosis acute respiratory distress syndrome, and COPD all include inflammation as a significant component . MicrobeBronchodilators and anti-inflammatory drugs are commonly used in the current therapeutic strategy for pulmonary disorders . The phoVitamins are nutritional molecules that are required for optimal health but are not produced by the human body. Cholecalciferol (Vitamin D) is classified as a vitamin, although it is a hormone. Vitamin D is thought to perform a function in bone and calcium homeostasis. Recent research has found that vitamin D can perform a variety of cellular processes, including cellular proliferation; differentiation; wound repair; healing; and regulatory systems, such as the immune response, immunological, and inflammation ,12. The We conducted a literature search using thesaurus terms and keywords in the PubMed, Embase, Science direct, Scopus, and Springer Library databases from database inception to 26 August 2020; the following search terms were used: vitamin D, asthma, lung cancer, bronchiectasis, pulmonary fibrosis, acute respiratory distress syndrome, and COPD. There were no language limitations. If data were not accessible, we contacted the authors of the papers. Additionally, we carefully reviewed the included articles’ references for the most recent reviews. We began with a preliminary screening of the titles and abstracts, followed by a full-text review. Exclusions included case reports, case series, duplicate reports, comments, and author reactions.The prohormone vitamin D must be converted to physiologically active molecules that link to nuclear receptors to regulate a variety of biological activities. The conventional and alternative vitamin D metabolic routes, as well as vitamin D metabolism’s hormonal control, are discussed in this section  13,14].,14.13,143 and ergocalciferol: vitamin D2 are both vitamin D isoforms. In plant fungi and yeasts, in the presence of UV light, ergosterol is converted into vitamin D2, which is consumed through a diet rich in plant-based foods like mushrooms. UV light in the epidermis of human skin converts 7-dehydrocholesterol to vitamin D3, which may also be found in animal sources like cod liver oil. Vitamin D from the food or in the epidermis of the skin attaches to a protein that binds to vitamin D (VDBP) in the bloodstream and is then transported to the hepatic system. Vitamin D 25-hydroxylase (CYP27A1 and CYP2R1) in the hepatic system converts vitamin D to the calcidiol that is 25(OH)D, which is the most common circulating type of vitamin D in the blood [2+ and MAPK signaling via direct peptide–peptide interactions with signaling molecules, a present intracellularly associated with certain phenotypic functions [Cholecalciferol: vitamin Dhe blood ,16. Calche blood . Calcitrhe blood . Calcitrunctions .A new vitamin D metabolism route involving CYP11A1 has just been discovered. Due to the discovery of CYP11A1 expression in GIT and the epidermis of the skin, it has come out as a novel metabolizing enzyme of calcitriol. 17(OH)D, 22(OH)D, and 20(OH)D are the most common vitamin D metabolites produced by CYP11A1. CYP11A1 hydroxylates these to produce 17,20,23 (OH)3D, 17,20(OH)2D, 20,22(OH)2D, and 20,23(OH)2D .+2 quantity, which is traced by receptors of Ca+2-sensing located on parathyroid cells. PTH increases calcitriol synthesis by stimulating renal CYP27B1 expression through methods such as enhanced regulation of the nuclear orphan receptor, the transcription dependent on NR4A2 or enhanced cAMP-dependent transcription [Calcitriol has a unique negative feedback process that closely controls the metabolism of vitamin D. The inactivating enzyme of vitamin D, CYP24A1, is one of the calcitriol–VDR–RXR complex’s strongest transcriptional targets. Around 250-bp and 150-bp upstream of the transcriptional initiation codon in the gene promoter of CYP24A1, two VDREs cause a significant stimulation of CYP24A1 by vitamin D, which could also promote CYP24A1 expression by attracting RNA polymerase II and histone H4 acetyl transferases to a region 50–70 kb downstream of the CYP24A1 gene. As a result, in the kidney calcitriol-driven CYP24A1, its expression may closely control the amounts of calcitriol and calcidiol ,23. Furtcription .The majority of individuals in the United States take less vitamin D than is advised. According to a 2015 to 2016 National Health and Nutrition Examination Survey (NHANES) analysis, the average daily vitamin D intake from foods and beverages was 5.1 micrograms  for men, 4.2 micrograms  for women, and 4.9 micrograms  for children aged 2–19 years. Indeed, according to the 2013–2016 NHANES data, 92% of males, more than 97% of women, and 94% of persons aged 1 year and older consumed less than the EAR of 10 mcg (400 IU) of vitamin D through foods and drinks . CertainInflammatory lung disease is a broad term for a group of diseases characterized by lung inflammation and an elevated neutrophil number. Acute and chronic inflammatory respiratory problems are both related with airway inflammation and are affected by a mix of ecological, genomic, and epigenetic factors .Inflammation is the body’s natural defensive mechanism for removing infections, toxins, and injured cells while also starting the process of healing. Pathogens or chemical exposure, allergens, contaminants, and irritants are the most common causes of airway inflammation. Toll-like receptors recognize pathogen-shared molecular processes and trigger inflammatory cells mediators such as TNF-α, chemokines, and cytokines such as interleukin 8 (IL-8) and NF-κB, which then generate growth factors to begin the corrective actions . TNF-α iMycobacterium tuberculosis activates the TLR1/2 heterodimer in macrophages, it causes the overexpression of VDR and CYP27B1, which culminates in the production of the antimicrobial peptide cathelicidin and the death of internalized M. tuberculosis. The differentiation of macrophages induced by TLR2/1 is linked to the antimicrobial pathway dependent on the vitamin D in this process, and IL-15 is involved. When the CYP27B1 levels rise, more 1,25(OH)2D3 is produced, which triggers VDR and causes target gene transcription through calcitriol responsive ingredients enshrined in the regulatory regions of the target genes of 1,25(OH)2D3 [Macrophages, an important cell type in the innate immune system, have been found to express VDR. When 5(OH)2D3 ,38. TLR 5(OH)2D3 . The mos5(OH)2D3 ,41.Vitamin D insufficiency has been related to numerous chronic illnesses in vulnerable populations exposed to airborne particles. The bioactive version of vitamin D, calcitriol, is important for immunological modulation . Many reAsthma is the most common noncommunicable illness, impacting millions of people’s health. Asthma is ranked 16th globally in terms of illnesses. Asthma affects about 300 million individuals worldwide, with an additional 100 million people predicted to get the disease by 2025. It is a multigenic inflammatory disease characterized by hyper-responsiveness of the lungs. Dyspnea, wheeze, chest discomfort, and coughing are all indications of asthma. Asthma sufferers encounter a variety of signs ranging from mild to serious, which might happen daily or only rarely. Asthma attacks can occur as the symptoms grow severe. Asthma usually begins in childhood, although it may strike anybody at any age ,52. AdulCOPD is among the leading reasons for death globally, and it is expected to be the third-largest reason for fatality by 2030. It is classified as an inflammatory disease of the pulmonary system by the GOLD guidelines. COPD is a severe pulmonary illness marked by continuous respiration difficulties, restricted airflow, and abnormal inflammation. Breathing difficulties and inadequate airflow to the lungs are caused by inflammation in the bronchial tube linings (obstructive bronchiolitis) and breathlessness owing to alveolar sac damage (emphysema) ,65. The Thus, vitamin D treatment in COPD patients with deficient vitamin D may be a useful and effective therapeutic strategy to avoid the disease from deteriorating. COPD exacerbation is connected to a high incidence of deficient vitamin D, although low vitamin D blood concentrations are not linked to susceptibility . VitaminLung cancer is the commonest reason of carcinoma-related death in both sexes across the globe. Non-small-cell lung cancer (NSCLC), which contributes to 85% of all cases of pulmonary carcinoma, and small-cell lung cancer (SCLC), which accounts for 15% of the cases, are the two primary types of lung cancer. The carcinoma of squamous cells, adenocarcinoma, and lung carcinoma of the large cells are the three types of NSCLC ,76. WhilVitamin D’s anticancer properties are considered to be triggered by calcitriol interacting with the VDR. Lung cancer cell growth is inhibited, apoptosis is promoted, and angiogenesis is reduced through these methods. A recent in vitro study found that calcitriol caused G0/G1 cell cycle blockage by downregulating cyclins that facilitate cell cycle entrance into the S phase. 1,25(OH)2D inhibits angiogenesis by inhibiting the production of VEGF, which is reported to stimulate endothelial cell excitation, recruitment, and multiplication. In lung cancer cells treated with 1,25(OH)2D, parathyroid hormone-related protein (PTHrP), MMP-9, and MMP-2 expression and synthesis are similarly decreased ,79. SincP. aeruginosa infection in a scientific investigation that had anti-inflammatory properties by lowering the level of IL-23 and IL-17A [Idiopathic pulmonary fibrosis (IPF) or pulmonary fibrosis is a long-term lung fibrotic infection characterized by a mix of hereditary and ecological variables. IPF can also be caused by cystic fibrosis. Cystic fibrosis (CF) is a fatal autosomal-recessive genetic condition that shortens the lifespan. It mostly affects the lungs, pancreas, and other organs, resulting in blockage and inflammation. A polymorphism in the CF transmembrane conductance regulator (CFTR) gene causes it . The FDAd IL-17A . Vitamind IL-17A . RAS oved IL-17A . UltimatPseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pneumonia, and Staphylococcus aureus); viruses ; fungi; and parasites can all cause pneumonia [Pneumonia is a serious pulmonary illness that has a high fatality rate. It leads to pus and fluid in filled alveoli, resulting in inflammation of the alveoli and adjacent tissue, resulting in a sluggish inhalation of oxygen. Indications such as sputum cough, high temperature, muscular tiredness, breathlessness, and others are seen as consequences. Bacteria  is the second-most well-known infectious illness, accounting for more than 2 million fatalities worldwide each year. A mild respiratory infection occurs during the early stages of tuberculosis, and a failure in identification and management at this point allows the infection to spread easily through sneezing and coughing . RegardlThe new coronavirus illness COVID-19), which was first discovered in late 2019 in Wuhan, China, is still spreading throughout the world, infecting more than 100 million people and killing approximately 2.4 million people in 221 nations around the globe. Due to the pandemic’s fast spread and very substantial fatality rates, particularly among vulnerable groups, controlling and preventing it has proven difficult ,106. The, which wVitamin D minimizes the incidences of viral infection and death in a variety of ways. Vitamin D works through three mechanisms to lower the chance of catching a cold: cellular natural immunity, adaptive immunity, and the physical barrier . Many inVitamin D is a necessary prohormone that is required for the maintenance of healthy bones and calcium levels. Vitamin D insufficiency results in hypocalcemia and bone mineralization abnormalities. Vitamin D has become a common alternative due to the rising awareness of vitamin D insufficiency and associated health issues, and its use has expanded significantly. Increased vitamin D supplementation by the general population and an increasing number of therapeutic dosage prescriptions (including extremely high doses) without medical supervision may result in an increased risk of exogenous hypervitaminosis D, commonly known as vitamin D toxicity .Hypervitaminosis D, along with hypercalcemia, occurs as a result of uncontrolled usage of megadoses of vitamin D or vitamin D metabolites. Hypervitaminosis D may develop in some clinical circumstances as a result of the use of vitamin D analogs (exogenous vitamin D toxicity). Hypervitaminosis D, in conjunction with hypercalcemia, may also be a symptom of excessive 1,25(OH)2D production in granulomatous diseases, lymphomas, and idiopathic infantile hypercalcemia (IIH). Exogenous vitamin D toxicity is often induced by the chronic usage of megadoses of vitamin D, not by excessive sun exposure or a diverse diet. The human body can control the amount of previtamin D (tachysterol and lumisterol) generated by ultraviolet B light in the skin. The vitamin D levels in a varied diet are often low, and the vitamin D fortification of food items is minimal . ExogenoVitamin D is an essential vitamin that influences the physiological activities, such as cellular differentiation and proliferation, host defense, immunological regulation, and inflammation, in addition to the usual bone and calcium homeostasis functions. Deficient vitamin D has been associated with different problems related to health, such as bone softening and skeletal abnormalities, as well as immunological disorders. A vitamin D deficit is also linked to inflammatory lung diseases, such as COVID-19, asthma, lung cancer, COPD, pulmonary and cystic fibrosis, pneumonia, and TB putting people with a vitamin D deficiency at a greater chance of developing respiratory diseases with substantial morbidity and fatality. As a result, the interrelationships among vitamin D and respiratory illnesses have sparked a new age of attention in supplementing as a cost-effective way to enhance world health. Furthermore, the clinical usage of vitamin D would aid in immunological sustenance, which would be an exciting future prospect.


In this study, Eudragit nanocapsules (NCs) containing nicotine were produced. Nicotine is the main alkaloid found in tobacco and has anti-inflammatory properties. NCs containing nicotine may be used as an adjuvant therapy in the treatment of inflammation in the central nervous system. (2) Methods: Nanocapsules were prepared by the interfacial deposition of the pre-formed polymer method and characterized in terms of zeta potential, diameter, polydispersity index, pH, encapsulation efficiency (EE), stability and sustained release profile. In vitro tests with the PC12 cell line were performed, such as MTT, LIVE/DEAD and ELISA assays, to verify their cytotoxic and anti-inflammatory effects. (3) Results: The nanocapsules presented satisfactory values of the characterization parameters; however, poor encapsulation was obtained for nicotine (8.17% ± 0.47). The in vitro tests showed that the treatment with nanocapsules reduced cell viability, which suggests that the Eudragit or the amount of polymer on top of the cells may be detrimental to them, as the cells were able to survive when treated with bulk nicotine. ELISA showed an increment in the expression of IL-6 and IL-1β, corroborating the hypothesis that NCs were toxic to the cells because of the increase in the levels of these pro-inflammatory cytokines. (4) Conclusions: This study demonstrates that NCs of Eudragit present toxicity. It is therefore necessary to improve NC formulation to obtain better values for the encapsulation efficiency and reduce toxicity of these nanodevices. Nanotechnology is an emerging interdisciplinary field that combines chemistry, physics, engineering and biology and has been extensively used in recent years in the health research area. It aims to create functional materials by controlling the matter in the nanometer scale, thus, producing materials with new chemical, physical and biological properties. It can be used particularly in the biomedical area to produce nanodevices or nanorobots, which are useful in diagnostic procedures; nanotechnology is also used to produce scaffolds or matrices utilized in regenerative medicine to mimic the extracellular matrix ,2. In adNanometrical devices of sustained release present several advantages compared to conventional administration forms, such as pills, injections, patches and sprays. Among these advantages, some of them include better therapeutic efficacy; reduction of side effects and thus, toxicity reduction; reduction of early inactivation of drugs; better targeting of the drug to the target tissue; ease of transposing biological barriers; and greater versatility, as it is possible to encapsulate lipophilic and hydrophilic compounds, among others . BesidesEudragit is the brand name for various polymethacrylate-based copolymers that includes anionic, cationic and neutral copolymers, based on methacrylic acid and methacrylic/acrylic esters or their derivatives . These pNanotechnology holds great promise in the development of new therapeutic strategies to diagnose, treat or even regenerate the nervous system in neurological disorders, such as multiple sclerosis, Parkinson’s disease, Alzheimer’s disease and spinal cord injury (SCI) . NanocapThe NF-kB pathway is crucial in the inflammatory response because of the induction of the expression of a series of pro-inflammatory genes, including those which encode chemokines and cytokines, such as the pro-inflammatory cytokines TNF-α, IFN-γ and IL-1β. Under physiologic conditions, nuclear transcription factor NF-kB induces the transcription of more than five hundred genes, regulating survival, activation and differentiation of cells of the innate immune system and inflammatory T cells, among other functions .Nicotine is the main alkaloid found in tobacco and, in the human organism, it has the capacity of binding to acetylcholine nicotinic receptors, which leads to the release of a variety of neurotransmitters such as noradrenaline, dopamine, acetylcholine, serotonin, GABA, glutamate and endorphins . FurtherBesides inhibiting the pro-inflammatory NF-kB pathway, various studies in the literature show the immunomodulatory effect of nicotine, suggesting that it can suppress T cell and B cell development and activation, and decrease T cell response, altering differentiation, phenotype and function of antigen presenting cells (APCs), such as macrophages and dendritic cells . Lee et In the present work, it was hypothesized that nicotine could be an adjuvant in the treatment of CNS diseases by reducing neuroinflammation. Therefore, a new strategy composed of polymeric nanocapsules containing nicotine has been produced and characterized. Additionally, the cytotoxic and anti-inflammatory effects of the nanocapsules containing nicotine were evaluated in vitro using the cellular neural model PC12 cell line.® RL, Evonik, São Paulo, SP, Brazil), 15 mg of nicotine ditartrate dihydrate  0.082 g of castor oil and 13.5 mL of acetone. Upon homogenization of the phases, the organic phase was poured into the aqueous phase, under vigorous magnetic stirring for 10 min. Following this, the excess solvent and water were evaporated in a rotary evaporator at 30 mbar and 40 °C bath heating in order to adjust the concentration of nicotine and reach the final volume of 5 mL of suspension.The nanocapsules were produced by the interfacial deposition of the pre-formed polymer method . InitialEmpty nanocapsule suspensions were prepared by the same method, without adding nicotine to the formula.The morphological characteristics of the prepared nanocapsules were evaluated by Transmission Electron Microscopy (TEM)  after preparation with negative contrast.The pH was determined at room temperature, using pH strips.The average size of the nanocapsules and the respective polydispersity index (PdI) were determined by the dynamic light scattering method . The samples were prepared by diluting the suspensions in sodium chloride (1 mM) at 1:100. Zeta potential was determined using the same equipment and the same samples.The nicotine content of the nanocapsules was determined by the High-Performance Liquid Chromatography (HPLC) method. The sample was diluted in 1 mL of methanol and incubated at room temperature overnight (18 h). Following this, the samples were filtered and injected in the HPLC system  for analysis. The following parameters were used: wavelength 259 nm, temperature 30 °C, injected volume 20 μm, mobile faze A water, mobile faze B methanol, and a flux 0.5 mL/min. For determining the encapsulation efficiency, the samples were filtered in microfiltration tubes composed of 0.1 μm pore membranes (MilliPore) by centrifugation  for 2 min at 6000 rpm, and the supernatants were collected. Nicotine content in the supernatants (free nicotine) was also determined by HPLC. The encapsulation efficiency was calculated as follows:The nicotine sustained release profile was analyzed using a vertical diffusion Franz cell, containing receptor and donator compartments and a cellulose membrane with 0.05 µm pores as a barrier. Phosphate buffered saline (PBS) was used as the receptor solution. This mixture was maintained at 37 °C during the entire time of the experiment. A volume of 500 µL of Eudragit nanocapsules containing nicotine was placed on top of the Franz cell, and, at the time intervals of 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h after the beginning of the experiment, 1 mL of receptor solution was collected and 1 mL of fresh receptor solution was replaced in the receptor compartment. The collected samples were analyzed in regard to the nicotine concentration by the HPLC method.A variety of nanocapsule suspensions were stored at 4 °C and protected from light. Immediately after preparation and after 7, 15, 30, and 90 days, the formulations were evaluated in terms of pH, particle diameter, polydispersity index (PdI) and zeta potential. Another set of suspensions were maintained at room temperature (25 °C) to evaluate if visible properties such as color and phase homogeneity would change in any manner.To assess the biosafety of the formulations, the nanocapsules used were subjected to a series of tests to evaluate their cytotoxicity and cellular viability. All the tests were performed in triplicate unless otherwise stated.2 at 37 °C.PC12 cells (ATCC CRL-1721), an established neural cell model derived from a pheochromocytoma of the rat adrenal medulla, were cultivated in culture flasks with DMEM high medium supplemented with 10% fetal bovine serum inactivated by heat, 5% horse serum and 1% penicillin/streptomycin, and maintained in an incubator with a humidified atmosphere with 5% COPrior to every in vitro test, the nanocapsule formulations were produced following the above method and sterilized by UV light for 30 min.In this experiment, for treatment with nicotine containing nanocapsules, two different concentrations of nicotine were used: 100 μM and 500 μM. Treatment of the control groups with empty nanocapsules was calculated based on the amount of Eudragit contained in the above concentrations.Cells cultivated on a tissue culture plate without nanocapsule treatment (control);Cells treated with 166 μg/mL of empty Eudragit nanocapsules  (EN100);Cells treated with 833 μg/mL empty Eudragit nanocapsules  (EN500);Cells treated with Eudragit nanocapsules containing 100 μM of nicotine (NNC100);Cells treated with Eudragit nanocapsules containing 500 μM of nicotine (NNC500);Cells treated with 100 μM of bulk nicotine dissolved in deionized water (NIC100);Cells treated with 500 μM of bulk nicotine dissolved in deionized water (NIC500);The cell experiment was divided into seven groups:Cell viability was evaluated by the Tetrazolium salt method (MTT), a colorimetric assay which uses the tetrazolium dye MTT 3--2,5-diphenyltetrazolium, which is reduced by the viable cells to its insoluble salt formazan, and which is purple in color. One thousand cells were plated and cultivated in 48-well plates, treated with nanocapsules and incubated for different time periods. After 1 and 7 days, the supernatants were removed and MTT reagent was added to the cell culture in the final concentration of 0.5 mg/mL. After 2 h of incubation at 37 °C, the MTT was removed and the formazan salts were dissolved in Dimethyl Sulfoxide (DMSO) and homogenized. Following this, the absorbance was measured by spectrophotometer at the wavelengths of 570 and 630 nm. The results were calculated by the difference of the values at the two wavelengths. Only viable cells are capable of reducing MTT, thus, elevating group absorbance after NC treatment.Cell viability was also evaluated by fluorescent staining with LIVE/DEAD kit , in accordance with the manufacturer’s protocol. This assay was performed 7 days after NC treatment.®, São Paulo, Brazil) in order to induce inflammation and then evaluate the NC anti-inflammatory effect.The IL-6, TNF-α and IL-10 cytokines were analyzed by the Enzyme-Linked Immunosorbent Assay (ELISA), following manufacturer recommendations  at 1 and 3 days after treatment with the nanocapsules. Prior to the nanocapsule treatment, the PC12 cells were stimulated with 10 mg/mL of LPS for 18 h .Appropriate statistical tests were employed to evaluate the significance level achieved, in accordance with the respective methodology, using the software GraphPad 7.0. Values are expressed as means and standard deviation. Means comparisons were performed by analysis of variance (ANOVA) followed by the Dunnett post-hoc test. The significance value adopted was 5%  analysis was performed. As shown in Freshly made Eudragit nanocapsules presented different ranges of pH, as demonstrated in Encapsulation efficiency was calculated, as described in the materials and methods section. The value obtained was of 8.17% ± 0.47 (mean and standard deviation).The nanocapsule suspensions were analyzed at five different time points: immediately after production—day 0, day 7, day 15, day 30 and day 90. The measured parameters were zeta potential (ZP), size (diameter), polydispersity index (PdI) and pH.Eudragit nanocapsules containing nicotine presented a sustained release profile, as illustrated in PC12 cells treated either with empty nanocapsules or nicotine containing nanocapsules exhibited significantly lower viability compared to the cells that received no treatment (control group), or those which received treatment with bulk nicotine, as shown in Cell viability at day 7 was also assessed by LIVE/DEAD assay , which cThe expression of cytokines TNF-α, IL-6 and IL-10 were evaluated by ELISA. IL-10 was not detected in our samples. At day one, IL-6 was significantly increased in the groups with higher empty nanocapsule concentration (EN500) and higher nicotine containing nanocapsule concentration (NNC500) . This inAt day one, TNF-α showed no significant difference in expression between the groups . HoweverIn this study, nanocapsules of Eudragit containing nicotine were successfully produced and further characterized. According to the literature, the parameters for particle diameter obtained were satisfactory, around 120 nm for empty nanocapsules and 130 nm for nicotine containing nanocapsules, and for nanocapsule size distribution, values close to 0.1, meaning that most of the capsules had similar sizes in the gaussian curve. Zeta potential also had optimal values (around +45 mV for both formulations). The results show that the addition of the drug did not significantly change the structure of the Eudragit nanocapsules. The NCs also presented a good stability of the formulation when stored for 90 days, without any parametric and macroscopic alterations. These nanocapsule properties are in accordance with studies from Dalcin (2019) and Katzer (2014) and colleagues, who prepared Eudragit nanocapsules with the same interfacial deposition of the pre-formed polymer method ,18. The However, for this formulation, a poor encapsulation efficiency for nicotine was achieved, which means a low amount of nicotine  was entrapped in the core of the nanocapsules. The encapsulation efficiency is highly dependent on the composition of the capsule core, such as the oil used, the characteristics of the drug encapsulated and the presence of surfactants that may facilitate the solubilization of the drug ,19. The After their entrance in the living organism, nanoparticles are usually targeted by opsonization and suffer phagocytosis by monocytes and macrophages, ending up in different organs such as the liver, spleen and lymph nodes . It is tIn this study, it was demonstrated that Eudragit can be toxic to PC12 cells, as it decreased their viability and stimulated the production of pro-inflammatory cytokines. PC12 cells are a good model for toxicology testing, especially when dealing with nanoscience applications. The aim of the capsules produced in this study is for treating neuropathological disease. The decreased viability was unexpected; however, nicotine concentration was probably not the issue, as the cells treated with bulk nicotine presented viability rates similar to the control group. Moreover, nicotine poly(lactic-co-glycolic) acid nanoparticles containing similar concentrations of nicotine (100 µM) proved to be neuroprotective in a dopaminergic neurons culture . It was To evaluate the anti-inflammatory effect of nanocapsules containing nicotine, the expression of three different cytokines were quantified by ELISA after treating the PC12 cells: IL-6, TNF-α and IL-10. Cytokines are essential for the inflammatory process as they induce the expression of additional cytokines, chemokines, nitric oxide and reactive oxygen species . The cytTo improve NC cytocompatibility, one possibility is to functionalize the polymer and, thus, the surface of the nanocapsule to improve cell-particle interaction. In addition, the fact that Eudragit is a non-biodegradable polymer could also influence this interaction. Fujii and colleagues (2001) argue that the exposure of epithelial cells to non-biodegradable particles induces a pro-inflammatory state and the production of cytokines . One stuAdditionally, it is not uncommon for nanoparticles to form a sediment on top of the adhered cells instead of remaining suspended in the culture media, thereby increasing, the density of particles at the cell surface. It is speculated that the nanoparticle weight on top of the cells can influence processes such as cellular uptake and cell death ,34, whicNevertheless, it is difficult to draw in vivo conclusions by in vitro tests, as the in vivo environment is much more complex: there is a greater number of cells and the presence of extracellular matrix and of circulatory systems, for example. Thus, it is vital to test this formulation in animal models, as understanding its pharmacokinetics is critical for gaining a better insight into its effects. In living organisms, nanoparticle toxicity is dependent on factors like organ distribution of the particle and retention times, which are mainly determined by nanoparticle features . A studyThereby, in future studies, it is our intention to improve the Eudragit nanocapsule formulation in order to achieve a better nicotine encapsulation efficiency value, lyophilizate the samples to increase their stability even further and reduce contamination risk; correct its pH to physiological values and, after further in vitro tests, perform in vivo experiments.In this study it was possible to produce Eudragit nanocapsules containing nicotine that were satisfactorily characterized regarding their zeta potential, diameter, polydispersity index, pH and sustained release profile. The nanocapsules also presented good stability over time when stored. However, a low encapsulation efficiency was obtained, and this has to be further analyzed and improved. In vitro treatment of PC12 cells with nanocapsules showed that Eudragit was toxic to cells as it decreased their viability. Moreover, Eudragit induced inflammation in these cells as it elevated the expression of pro-inflammatory cytokines. More studies are therefore needed to better understand the cytotoxicity exhibited by this polymer.


Background: Rapid eye movement sleep deprivation (REMSD) and fluoxetine affect depression, yet the detailed molecular mechanisms were not clear.Methods: Rat depression chronic unpredictable stress was constructed, and the body weight of rats was measured. The efficacy of REMSD and fluoxetine on the pleasure experience, exploration, and cognition of rats with depression was determined by the Sucrose preference test, the open field test, and Morris water task, respectively. The effects of REMSD and fluoxetine on depression-induced damage and apoptosis in rat hippocampi were detected using hematoxylin–eosin staining and terminal transferase-mediated biotin 2′-deoxyuridine, 5′-triphosphate nick end labeling. A1 adenosine receptor content was measured by immunohistochemistry. Relative expressions of the A1 adenosine receptor, proteins related to apoptosis , phosphoinositide 3-kinase, P38 mitogen-activated protein kinase, cFos, and adenosine deaminase RNA specific two were quantified by quantitative real-time polymerase chain reaction and Western blot as needed.Results: Depression decreased rat weight. REMSD combined with fluoxetine increased body weight, prompted rat behavior, alleviated depression-induced damage, attenuated apoptosis, and promoted A1 adenosine receptor level in rat hippocampi. Furthermore, the combined therapy upregulated expressions of A1 adenosine receptor, B-cell lymphoma 2, and phosphoinositide 3-kinase but downregulated those of B-cell lymphoma 2-associated X protein, P38 mitogen-activated protein kinase, cFos, and adenosine deaminase RNA specific 2 in the hippocampi of rats with depression.Conclusion:REMSD combined with fluoxetine protected rats against depression-induced damage and apoptosis in the hippocampus via the A1 adenosine receptor, providing a possible treatment strategy for depression. Depression has been recognized as one of the most significant causes of emotional suffering and a factor affecting the morbidity of several medical disorders , 2. PatiVarious researches on epidemiology unveiled the relation of sleep duration with health-related conditions, such as cardiovascular health, mental disorders, and mortality –7. Also,via enrolling the four G protein-coupled receptors   . A1 aden and A3) . Several and A3) . Meanwhi and A3) . HoweverAll the animal experiments were performed in line with the guidelines of the China Council on Animal Care and Use. The Committee of Experimental Animals of Zhejiang Chinese Medical University has approved our current study (JSK2019091001). Every effort was made to minimize pain and discomfort of the animals. All animal experiments were performed at Zhejiang Chinese Medical University.n = 50) were purchased from the Experimental Animal Center of Henan Province [license number: SXCK (Henan) 2012–0011] and housed in pathogen-free cages (two/cages) under the conditions as follows: 12-h day/light cycle, 22–26°C, and 50–60% humidity. Before our study, all rats were allowed to have free access to a standard experimental diet and sterile tap water.In the current study, male Sprague-Dawley rats : control, CUS, CUS + REMSD, CUS + fluoxetine, and CUS + REMSD + fluoxetine groups. Rats in the control group were housed normally without any treatment, and rats in the remaining groups were subjected to depression model construction as described in a prior study  was obtained from Sigma-Aldrich , and the rats were randomly assigned to five groups , whereas rats in the remaining groups were intraperitoneally injected with fluoxetine (5 mg/kg/day) once a day for 28 days with or without REMSD, and their body weight was measured again on days 0 and 28 after the treatment with REMSD and fluoxetine.via inhalation of 5% isoflurane , and hippocampi samples were collected and stored at 4°C after washing with phosphate-buffered saline . The experimental design is outlined in Finally, all the rats were killed The sucrose preference test was performed to determine the capability of rats to experience pleasure in line with a prior study . In detaA previous study showed that the open field test could be used to analyze the anxiety-like and locomotor behavior of the rats , 25. In For the open field test, the acrylic box was cleaned using 5% ethanol  before introducing rats, and then rats were placed at the corner of the open field facing the wall. The behavior of each rat was recorded for 5 min, and the data from the postrecording of the horizontal movement distance, immobility time , retention within 5 min, and numbers of rats entering the central region were analyzed. Immobility time represented motility, and higher scores of immobility time indicated severe depression.Morris water task was performed on day 28 post-REMSD, and fluoxetine treatment was used in accordance with previous research to test the cognition ability of rats . In our For hematoxylin–eosin (H&E) staining, hippocampus tissue of rats was harvested with caution, fixed in 4% paraformaldehyde , and finally embedded in pre-dissolved paraffin . Samples were resected, stained by an H&E Stain Kit , and analyzed under an inverted light microscope  under × 100 and × 200 magnifications.Terminal transferase-mediated biotin 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) assay was performed to evaluate apoptosis of cells with a TUNEL assay kit  as previously described . The hipn = 10.The collected hippocampus tissue was fixed in 4% paraformaldehyde solution overnight and then sliced into sections with a thickness of 5 μm, followed by embedding in paraffin for staining. The paraffin sections were stained with anti-A1 adenosine receptor antibody  at 4°C overnight and then stained using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody  at room temperature and a 3,3′-diaminobenzidine kit for 15 min. The nuclei were counterstained using hematoxylin , and the positive cells in five randomly chosen fields were counted and observed with a stereomicroscope. Here, the sample size was TM SYBR Green One-Step qRT-PCR kit  in the AriaMx real-time PCR System  under the following conditions: at 95°C for 10 min, and 40 cycles of at 95°C for 20 s and at 60°C for 30 s, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. Primer sequences are listed in −ΔΔCT method  was used for extracting total RNA from hippocampus tissues based on the manuals of the manufacturer. The extracted RNA was preserved at −80°C, and the concentration was determined using a NanoDrop spectrometer . Of total RNA, 1 μg was synthesized into complementary DNA using a complementary DNA synthesis kit . Quantitative real-time polymerase chain reaction (QRT-PCR) was conducted using a BeyoFastT method . Here, tn = 10.Protein expressions were measured by Western blot based on a previous study . After tn = 10). Statistics were analyzed using Graphpad prism 8.0 software . Comparison between two groups was performed using the Student's t-test. Comparisons between multiple groups were performed using a one-way analysis of variance, followed by a Dunnett's post-hoc test. The data were determined to be statistically significant when P-value < 0.05.The data were expressed as mean ± standard deviation , which suggested that depression decreased the rat body weight.To discover the possible effects of depression on rats, the rats were first exposed to CUS for the construction of depression, and their body weight was measured on days 1, 7, 14, 21, and 28 after model construction. As shown in P < 0.001). However, the body weight of rats was increased after treatment with REMSD or fluoxetine alone, and it was further enhanced by co-treatment with REMSD and fluoxetine (P < 0.05), suggesting that REMSD and fluoxetine treatment could promote the body weight of rats with depression.After depression model construction by CUS and treatment with REMSD and fluoxetine, the body weight of rats was measured, and their capability of pleasure experience, exploration, and cognition was determined by the Sucrose preference test, the open field test, and Morris water task, respectively. Meanwhile, the locomotor activity of rats was monitored as well. In P < 0.001). However, REMSD or fluoxetine treatment increased the consumed volume of Sucrose and the distance of horizontal movement, retention time for 5 min, and times entering the central region but decreased the immobility time, and the effect was further enhanced by co-treatment with REMSD and fluoxetine (P < 0.001). Meanwhile, from the results of the Morris water task, we observed a high latency but reduced times of space exploration after the model construction of depression (P < 0.001). However, after REMSD or fluoxetine treatment, the latency was reduced, but the time of space exploration was increased, and the effects were further promoted by co-treatment of REMSD and fluoxetine (P < 0.05).The results from the Sucrose preference test and the open field test showed that after construction of the depression model, the consumed volume of Sucrose, the distance of horizontal movement, retention time for 5 min, and times entering the central region were decreased, whereas the immobility time was increased . However, an opposite result was found after REMSD or fluoxetine treatment alone (P < 0.05), and in comparison, the combined therapy of REMSD and fluoxetine further promoted the effects of REMSD and fluoxetine treatments on upregulating the expressions of the A1 adenosine receptor, Bcl-2, and PI3K and suppressing those of Bax, P38 MAPK, cFos, and ADAR2 in the hippocampi of rats with depression (P < 0.001).To further confirm the effects of the combination therapy of REMSD and fluoxetine on rats with depression, the expressions of the A1 adenosine receptor, apoptosis- and PI3K/P38 MAPK axis-related proteins, cFos, and ADAR2 were measured by Western blot and qRT-PCR. In Cognitive impairment is a common, often persistent, symptom of MDD. The ultimate goal of treatment in depression is a full functional recovery, and evaluating patients with cognitive dysfunction for cognitive impairment and treatment selection should lead to improved functional outcomes . At presvise versa, thereby showing the importance of identifying bidirectional risk factors for anxiety and depressive disorders  have been unveiled to have regulatory effects on various biological functions and play a role in diverse conditions, such as cerebral and cardiac ischemia, immune and inflammatory disorders, and cancer . It has We also focused on investigating possible mechanisms through which SD in combination with fluoxetine may provide better protection when compared with individual treatments. Fluoxetine is a serotonin reuptake blocker that suppresses REM sleep in mammals . The preThis study design has limitations. Selective REMSD is not feasible in humans, which decreases the external validity and translational potential of our paper . Besides, accumulating evidence has indicated the role of REMSD as the cause and effect of mania . Thus, tIn conclusion, we provided evidence of the efficacy of the combined therapy of REMSD and fluoxetine on the treatment of depression and its interaction with the A1 adenosine receptor. Our present study also provided novel evidence of the protective effect of the combined therapy on the hippocampus against depression-induced damage and apoptosis. It is hoped that our current study can bring further insights into the pathogenesis of depression and provide potential methods for the prevention and treatment of depression in clinical practice in the future.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Committee of Experimental Animals of Zhejiang Chinese Medical University.XJ and SW: substantial contributions to conception and design. PY, CZ, XH, JD, and ZT: data acquisition and data analysis and interpretation. XJ and SW: drafting the article or critically revising it for important intellectual content. XJ, SW, PY, CZ, XH, JD, and ZT: final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved. 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.


Leptospira interrogans and can spread this bacterium to the environment. Although Canicola is responsible for determining chronic disease in dogs, when affected by incidental serogroups such as Icterohaemorrhagiae, acute disease may occur with a predominance of clinical signs with hepatic and renal lesions. In endemic areas, it is a serious public health problem, as dogs can become asymptomatic carriers of leptospires in the urine, characterizing a risk in the context of zoonotic leptospirosis. Thus, this study aims to estimate the incidence and duration of the elimination of leptospires in the urine of dogs, taking another step from a previous study of our group, by a longitudinal, long-term and molecular approach. A total of 125 dogs without signs of leptospirosis were included in the study. Of the 125 dogs, 62 were PCR-positive ) throughout the study. Most dogs were shedding the Icterohaemorrhagiae serogroup in urine, which was unexpected, since the animals remained clinically asymptomatic during the study. Although the fact that asymptomatic dogs eliminate leptospires is not new, the extent of this fact and that the strain is virulent is impressive, with an impact on public health that cannot be overlooked.Dogs are known as hosts of Leptospira interrogans and can spread this bacterium to the environment. Although Canicola is responsible for determining chronic disease in dogs, when affected by incidental serogroups such as Icterohaemorrhagiae, acute disease may occur with a predominance of clinical signs with hepatic and renal changes. In endemic areas, it is a serious public health problem. Thus, this study aims to estimate the incidence and duration of elimination of leptospires in the urine of dogs, taking another step from a previous study of our group, by a longitudinal, long-term and molecular approach. (2) Methods: A total of 125 dogs without apparent symptoms of leptospirosis were included in the study. The dogs were all PCR-negative and seronegative at the beginning of the study. Blood samples were collected for hematological examinations and urine for amplification of the lipL32 gene by PCR at five different time points during one year. (3) Results: Out of the 125 dogs, 62 became lipL32 PCR-positive ) at some point during the study, distributed as follows: at day 0, all negative; day 90, 18/125 ); day 180, 18/125 ); day 270, 12/125 ); and day 365, 14/125 ). Out of the 62 amplicons, 22 were sequenced, targeting a short region of secY gene. Of these, 20 (90.9%) were identical to the L. interrogans serovar Icterohaemorrhagiae, while two (9.1%) were Leptospira noguchii. (4) Conclusions: The fact that the leptospires of the Icterohaemorrhagiae serogroup were characterized was unexpected, since the animals remained clinically asymptomatic during the study. The fact that asymptomatic dogs shed leptospires is not new, but the extent of this fact and the characterized strain is impressive, with an impact on public health that cannot be overlooked.(1) Background: Dogs are known as hosts of  Leptospira. It is characterized by being the most common zoonosis, which is responsible for more than 1 million cases worldwide, with 60,000 deaths per year [Leptospira interrogans serogroup Canicola and can spread this serovar to the environment [Leptospirosis is an infectious disease of worldwide distribution, caused by pathogenic species of the genus ironment ,3. In adironment ,5.Canine leptospirosis is widely described worldwide ,6. LeptoConsidering this, this study aims to estimate the incidence and duration of the elimination of leptospires in the urine of dogs, taking another step from a previous study of our group , by a loThe study was conducted after approval by the Ethics Committee of Universidade Federal Fluminense (CEUA no 3778190419).Leptospira interrogans serovars Autumnalis (Akiyami A), Bratislava (Jez-Bratislava), Bataviae (Van Tienen), Canicola (Hond Utrecht IV), Grippotyphosa (Moska V), Icterohaemorrhagiae (RGA), Copenhageni (M 20) and Pomona (Pomona).A total 125 dogs, no matter the sex, aged 2–9 years were studied, with no apparent symptoms of leptospirosis or any other infectious disease. The dogs were all from a shelter located in the same endemic region of our previous study, the municipality of São Gonçalo, Rio de Janeiro State [The shelter was located in a private urban area, surrounded and divided into covered pens or kennels, and the center of the enclosure was without a covered area. All dogs were mixed, having free access to the kennels and a common area for all. The shelter had a very poor infrastructure, without painting and featuring unfinished work, but was always clean. Rodent control was performed every six months. The entire floor was cemented, and the animals were separated individually or in groups of up to three animals in the pens, but all lived together. Dogs were fed once a day. The shelter received regular visits and assistance from a veterinarian, as well as health surveillance.® Biochemical Analyzer, Bioplus Products for Laboratórios Ltda, Cep: 06407-000 Barueri, Sao Paulo, Brazil). In these tests, the serum activity of alanine amino transferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase was analyzed, in addition to serum dosages of urea and creatinine, using commercial kits  according to the manufacturer’s instructions. Urine (for PCR) was also collected from all animals monthly. Urine was collected by ultrasound-guided cystocentesis. Prevalences were calculated using an error of 5% and 95% confidence intervals (CIs) with OpenEpi.All the dogs were followed up and evaluated for 365 days. Sampling occurred at days 0, 90, 180, 270 and 365. At each collection, visits were always made by the same veterinarian for a complete clinical examination of the body condition, including variables such as temperature, hydration, mucosal color, tissue perfusion and cardiorespiratory sounds. In addition, monthly blood tests were carried out, whereupon five mL of blood was collected from the animals selected in a tube with and without an ethylenediamino tetra-acetic acid (EDTA) anticoagulant. With the blood samples, a complete blood count was performed , as were biochemical tests . The total volume of each sample was analyzed by agarose gel electrophoresis (2%), stained with gel red and the DNA bands were visualized under ultraviolet light. The expected size of the amplicon was approximately 240 bp, varying slightly between the different species of Leptospira [Urine samples were centrifugated to increase the chance of recovering leptospires. DNA from urine samples was extracted by the commercial Wizard SV Genomic DNA Purification System kit following the manufacturer’s instructions . PCR assay targeted the lipL32 gene (present majority in pathogenic leptospires) using the primers LipL32-45F (5′-AAG CAT TAC CGC TTG TGG TG-3′) and LipL32-286R (5′-GAA CTC CCA CAG CGA TT-3) in a final volume of 25 µL following conditions described in  (p. 7). To confirm the identity of the amplicons, PCR products were selected and subjected to nucleotide sequencing based on the secY gene, after nested PCR following conditions described in . AmplicoL. interrogans serovar Icterohaemorrhagiae (99.2–100% identity) and 2/22 as Leptospira noguchii (both 99.7% identity). The samples identified in the sequencing as L. interrogans were found on day 365 (n = 7), day 270 (n = 3), day 180 (n = 6) and day 90 (n = 4). Regarding L. noguchii, one sample was found on day 365 and the other on day 270.No clinical or hematological/biochemical alterations were observed throughout the study. Out of the 125 dogs, 62 became lipL32-PCR positive ) at some point in the study, distributed as follows: at day 0, all negative; day 90, 18/125 ); day 180, 18/125 ); day 270, 12/125 ); and day 365, 14/125 ). Eight dogs presented positive more than once, but not consecutively. Of the 62 obtained amplicons, 22 yielded good sequences of the secY gene. Pairwise/Blast/NCBI comparisons with the GenBank secY gene dataset identified 20/22 of them as L. interrogans and L. noguchii species identification with high support values . Moreover, L. interrogans sequences from the present study clustered together with strains from the serovar Icterohaemorrhagiae, most of them from the same Brazilian geographical region. Similarly, L. noguchii species from the present study grouped with sequences from South America. The nucleotide sequences were deposited in GenBank under accession numbers L. interrogans: MW196272–MW196291, L. noguchii: MW196292–MW196293  of the sequenced amplicons, since there are not many studies showing this leptospiral species in dogs. It has been reported in different hosts, such as wild animals [L. noguchii was found in canines, suggesting a correlation between the infection of domestic animals and the increasing incidence of human cases [L. noguchii in southern Brazil, where three strains of L. noguchii were isolated, including two in humans and one in a dog, leading to severe clinical disease in the humans and the dog’s death [Another interesting outcome of the study was the characterization of n 9.1% 2/ of the s animals , mainly  animals . In a stan cases . It was ’s death . TherefoThe most unexpected outcome of the study was the molecular evidence that these asymptomatic dogs were shedding an Icterohaemorrhagiae strain, most likely L1-130, a highly virulent strain, important in the incidental infections of dogs worldwide . It is cIcterohaemorrhagiae strains are known to be highly virulent to dogs and humans, leading to severe clinical disease and very often death. The fact that the dogs of the present study became maintenance hosts of this strain without developing clinical disease was highly unexpected, and the reason why it happened remains to be elucidated, which indicates that the agent–host–environment relationship still requires a lot of research to be fully understood.The shedding of leptospires by asymptomatic dogs in endemic areas is probably much more common than expected and has been largely neglected. A high number of naturally exposed animals became shedders during the study, and a highly virulent strain of serogroup Icterohaemorrhagiae was being shed by the majority of the dogs. The impact of these outcomes on public health cannot be neglected.