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Parkinson's Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2's kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.
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[This corrects the article DOI: 10.3389/fimmu.2020.611522.].
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[This corrects the article DOI: 10.3389/fimmu.2022.825426.].
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Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
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Repetição de Anquirina , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas rab de Ligação ao GTP , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Células HEK293 , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Fosforilação , Microscopia Crioeletrônica , Ligação ProteicaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.
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Doença de Parkinson , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Microscopia Crioeletrônica , Fosforilação , MutaçãoRESUMO
The objective is to assess the circulating lipidome of children with obesity before and after lifestyle intervention and to compare the data to the circulating lipidome of adults with obesity before and after bariatric surgery. Ten pediatric (PE) and thirty adult (AD) patients with obesity were prospectively recruited at a referral single center. The PE cohort received lifestyle recommendations. The AD cohort underwent bariatric surgery. Clinical parameters and lipidome were analyzed in serum before and after six months of metabolic intervention. The abundance of phosphatidylinositols in the PE cohort and phosphatidylcholines in the AD significantly increased, while O-phosphatidylserines in the PE cohort and diacyl/triacylglycerols in the AD decreased. Fifteen lipid species were coincident in both groups after lifestyle intervention and bariatric surgery. Five species of phosphatidylinositols, sphingomyelins, and cholesteryl esters were upregulated. Eight species of diacylglycerols, glycerophosphoglycerols, glycerophosphoethanolamines, and phosphatidylcholines were downregulated. Most matching species were regulated in the same direction except for two phosphatidylinositols: PI(O-36:2) and PI(O-34:0). A specific set of lipid species regulated after bariatric surgery in adult individuals was also modulated in children undergoing lifestyle intervention, suggesting they may constitute a core circulating lipid profile signature indicative of early development of obesity and improvement after clinical interventions regardless of individual age.
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Obesidade Infantil , Humanos , Adulto , Criança , Projetos Piloto , Lipidômica , Esfingomielinas , Fosfatidilcolinas/metabolismo , FosfatidilinositóisRESUMO
The partial remission (PR) phase of type 1 diabetes (T1D) is an underexplored period characterized by endogenous insulin production and downmodulated autoimmunity. To comprehend the mechanisms behind this transitory phase and develop precision medicine strategies, biomarker discovery and patient stratification are unmet needs. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression and modulate several biological processes, functioning as biomarkers for many diseases. Here, we identify and validate a unique miRNA signature during PR in pediatric patients with T1D by employing small RNA sequencing and RT-qPCR. These miRNAs were mainly related to the immune system, metabolism, stress, and apoptosis pathways. The implication in autoimmunity of the most dysregulated miRNA, miR-30d-5p, was evaluated in vivo in the non-obese diabetic mouse. MiR-30d-5p inhibition resulted in increased regulatory T cell percentages in the pancreatic lymph nodes together with a higher expression of CD200. In the spleen, a decrease in PD-1+ T lymphocytes and reduced PDCD1 expression were observed. Moreover, miR-30d-5p inhibition led to an increased islet leukocytic infiltrate and changes in both effector and memory T lymphocytes. In conclusion, the miRNA signature found during PR shows new putative biomarkers and highlights the immunomodulatory role of miR-30d-5p, elucidating the processes driving this phase.
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Introduction: The Covid-19 pandemic soon became an international health emergency raising concern about its impact not only on physical health but also on quality of life and mental health. Rare diseases are chronically debilitating conditions with challenging patient care needs. We aimed to assess the quality of life and mental health of patients with rare diseases in Spain, with a special focus on inherited metabolic disorders (IMD). Methods: A prospective case-control study was designed, comparing 459 patients suffering from a rare disease (including 53 patients with IMD) and 446 healthy controls. Quality of life (QoL) and mental health were assessed using validated scales according to age: KINDL-R and the Pediatric Symptom Checklist (PSC) for children and the WhoQoL-Bref questionnaire, GAD and PHQ-9 in adults. Results: First, children and adults (but not adolescents) with IMD showed greater psychological effects than controls (p = 0.022, p = 0.026 respectively). Second, when comparing QoL, only adult patients with IMD showed worse score than controls (66/100 vs 74,6/100 respectively, p = 0.017). Finally, IMD had better quality of life than other rare neurological and genetic diseases (p = 0.008) or other rare diseases (p < 0.001 respectively) but similar alteration of the mental status. Conclusions: Our data show that the pandemic had a negative impact on mental health that is more evident in the group of patients with IMD. Young age would behave as a protective factor on the perception of QoL. Furthermore, patients with IMD show a better QoL than other rare diseases.
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The kinetics of light emission in halide perovskite light-emitting diodes (LEDs) and solar cells is composed of a radiative recombination of voltage-injected carriers mediated by additional steps such as carrier trapping, redistribution of injected carriers, and photon recycling that affect the observed luminescence decays. These processes are investigated in high-performance halide perovskite LEDs, with external quantum efficiency (EQE) and luminance values higher than 20% and 80 000 Cd m-2 , by measuring the frequency-resolved emitted light with respect to modulated voltage through a new methodology termed light emission voltage modulated spectroscopy (LEVS). The spectra are shown to provide detailed information on at least three different characteristic times. Essentially, new information is obtained with respect to the electrical method of impedance spectroscopy (IS), and overall, LEVS shows promise to capture internal kinetics that are difficult to be discerned by other techniques.
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Background: Betamethasone, a glucocorticoid used to induce lung maturation when there is a risk of preterm delivery, can affect the immune system maturation and type 1 diabetes (T1D) incidence in the progeny. It has been described that prenatal betamethasone protects offspring from experimental T1D development. The main aim of this study was to evaluate the possible association between betamethasone prenatal exposure and T1D in humans. Research Design and Methods. A retrospective case-control study with a total of 945 children, including 471 patients with T1D and 474 healthy siblings, was performed. Participants were volunteers from the Germans Trias i Pujol Hospital and DiabetesCero Foundation. Parents of children enrolled in the study completed a questionnaire that included questions about weeks of gestation, preterm delivery risk, weight at birth, and prenatal betamethasone exposure of their children. Multiple logistic regression was used to detect the association between betamethasone exposure and T1D. Results: We compared T1D prevalence between subjects prenatally exposed or unexposed to betamethasone. The percent of children with T1D in the exposed group was 37.5% (21 of 56), and in the unexposed group was 49.52% (410 of 828) (p = 0.139). The percentage of betamethasone-treated subjects with T1D in the preterm group (18.05%, 13 of 72) was significantly higher than that found in the control group (12.5%, 9 of 72) (p = 0.003). The odds ratio for T1D associated with betamethasone in the univariate logistic regression was 0.59 (95% confidence interval, 0.33; 1.03 [p = 0.062]) and in the multivariate logistic regression was 0.83 (95% confidence interval, 0.45; 1.52 [p = 0.389]). Conclusions: The results demonstrate that the prenatal exposure to betamethasone does not increase T1D susceptibility, and may even be associated with a trend towards decreased risk of developing the disease. These preliminary findings require further prospective studies with clinical data to confirm betamethasone exposure effect on T1D risk.
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Betametasona/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/diagnóstico , Adulto , Betametasona/metabolismo , Betametasona/uso terapêutico , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Diabetes Mellitus Tipo 1/epidemiologia , Feminino , Alemanha/epidemiologia , Glucocorticoides/efeitos adversos , Glucocorticoides/metabolismo , Glucocorticoides/uso terapêutico , Humanos , Pediatria/métodos , Pediatria/estatística & dados numéricos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/epidemiologia , Efeitos Tardios da Exposição Pré-Natal/etiologia , Estudos RetrospectivosRESUMO
The partial remission (PR) phase, a period experienced by most patients with type 1 diabetes (T1D) soon after diagnosis, is characterized by low insulin requirements and improved glycemic control. Given the great potential of this phase as a therapeutic window for immunotherapies because of its association with immunoregulatory mechanisms and ß-cell protection, our objective was to find peripheral immunological biomarkers for its better characterization, monitoring, and prediction. The longitudinal follow-up of 17 pediatric patients with new-onset T1D over one year revealed that, during the PR phase, remitter patients show increased percentages of effector memory (EM) T lymphocytes, terminally differentiated EM T lymphocytes, and neutrophils in comparison to non-remitter patients. On the contrary, remitter patients showed lower percentages of naïve T lymphocytes, regulatory T cells (TREG), and dendritic cells (DCs). After a year of follow-up, these patients also presented increased levels of regulatory B cells and transitional T1 B lymphocytes. On the other hand, although none of the analyzed cytokines (IL-2, IL-6, TGF-ß1, IL-17A, and IL-10) could distinguish or predict remission, IL-17A was increased at T1D diagnosis in comparison to control subjects, and remitter patients tended to maintain lower levels of this cytokine than non-remitters. Therefore, these potential monitoring immunological biomarkers of PR support that this stage is governed by both metabolic and immunological factors and suggest immunoregulatory attempts during this phase. Furthermore, since the percentage of TREG, monocytes, and DCs, and the total daily insulin dose at diagnosis were found to be predictors of the PR phase, we next created an index-based predictive model comprising those immune cell percentages that could potentially predict remission at T1D onset. Although our preliminary study needs further validation, these candidate biomarkers could be useful for the immunological characterization of the PR phase, the stratification of patients with better disease prognosis, and a more personalized therapeutic management.
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Diabetes Mellitus Tipo 1 , Biomarcadores/metabolismo , Criança , Citocinas/uso terapêutico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/terapia , Humanos , Insulina/uso terapêutico , Interleucina-17 , Indução de RemissãoRESUMO
OBJECTIVES: S100A4 has been recently identified as an adipokine associated with insulin resistance (IR) in adult subjects with obesity. However, no data about its levels in children with obesity and only a few approaches regarding its potential mechanism of action have been reported. To obtain a deeper understanding of the role of S100A4 in obesity, (a) S100A4 levels were measured in prepubertal children and adult subjects with and without obesity and studied the relationship with IR and (b) the effects of S100A4 in cultured human adipocytes and vascular smooth muscle cells (VSMCs) were determined. METHODS: Sixty-five children (50 with obesity, age 9.0 ±1.1 years and 15 normal weight, age 8.4 ±0.8 years) and fifty-nine adults (43 with severe obesity, age 46 ±11 years and 16 normal weight, age 45 ±9 years) were included. Blood from children and adults and adipose tissue samples from adults were obtained and analysed. Human adipocytes and VSMC were incubated with S100A4 to evaluate their response to this adipokine. RESULTS: Circulating S100A4 levels were increased in both children (P = .002) and adults (P < .001) with obesity compared with their normal-weight controls. In subjects with obesity, S100A4 levels were associated with homeostatic model assessment-insulin resistance (HOMA-IR) in adults (ßstd = .42, P = .008) but not in children (ßstd = .12, P = .356). Human adipocytes were not sensitive to S100A4, while incubation with this adipokine significantly reduced inflammatory markers in VSMC. CONCLUSIONS: Our human data demonstrate that higher S100A4 levels are a marker of IR in adults with obesity but not in prepubertal children. Furthermore, the in vitro results suggest that S100A4 might exert an anti-inflammatory effect. Further studies will be necessary to determine whether S100A4 can be a therapeutic target for obesity.
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Type 1 diabetes (T1D) is a chronic metabolic disease characterized by the autoimmune destruction of ß-cells in the pancreatic islets. T1D is preceded by islet-specific inflammation led by several immune cells. Among them, natural killer (NK) cells are emerging as important players in T1D development. Human NK cells are characterized by CD56 and CD16 expression, which allows classifying NK cells into four subsets: 1) CD56dimCD16+ or effector NK cells (NKeff); 2) CD56brightCD16- or regulatory NK cells (NKreg); 3) intermediate CD56brightCD16+ NK cells; and 4) CD56dimCD16- NK cells, whose function is not well determined. Since many studies have shown that T1D progression is associated with changes in various immune cell types, we hypothesize that the kinetics of NK cell subsets in the blood could correlate with different stages of T1D. To that aim, pediatric patients newly diagnosed with T1D were recruited, and peripheral NK cell subsets were analyzed by flow cytometry at several disease checkpoints: disease onset, partial remission (PR), 8 months (for non-remitters), and 12 months of progression. Our results showed that total NK cells and their four subsets are altered at the early stages of T1D. A decrease in the counts and percentage of total NK cells and NKeff cells at the different disease stages was found when compared to controls. These results suggest the extravasation of these cells into the islets at disease onset, which is maintained throughout the follow-up. By contrast, NKreg cells increased during the early stages after T1D onset, and both intermediate NK cells and CD56dimCD16- NK cells diminished at the PR stage, which might reflect the immunoregulatory attempts and could be candidate biomarkers for this stage. Also, CD56dimCD16- NK cells increased during T1D progression. Finally, changes in CD16 expression were identified in the different T1D stages, highlighting a CD16 expression reduction in total NK cells and NKeff cells 1 year after diagnosis. That may reflect a state of exhaustion after multiple cell-to-cell interactions. Altogether, our preliminary data provide a longitudinal picture of peripheral NK cell subpopulations during the different T1D stages, which could be potential candidate biomarkers indicators of disease progression.
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Diabetes Mellitus Tipo 1/imunologia , Células Matadoras Naturais/imunologia , Pâncreas/imunologia , Adolescente , Fatores Etários , Biomarcadores/metabolismo , Antígeno CD56/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/terapia , Progressão da Doença , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunofenotipagem , Células Matadoras Naturais/metabolismo , Estudos Longitudinais , Masculino , Pâncreas/metabolismo , Pâncreas/patologia , Fenótipo , Projetos Piloto , Receptores de IgG/metabolismo , Indução de Remissão , Fatores de Tempo , Resultado do TratamentoRESUMO
Type 1 diabetes (T1D) is prompted by defective immunological tolerance, an event in which dendritic cells (DCs) are crucial as immune response orchestrators. In fact, they contribute to maintaining tolerance to self-antigens, but they can also prompt an immunogenic response against them, leading to autoimmunity. Countless factors can potentially impact on the proper functionality of the DCs, which range from altered subset distribution, impaired phagocytic function to abnormal gene expression. Moreover, in T1D, metabolic dysregulation could impair DC functions as well. Indeed, since T1D clinical course is likely to be more aggressive in children and adolescents and entails severe dysglycemia, the aim of this study was to analyze circulating DCs subpopulations in pediatric T1D at different stages, as well as to characterize their phagocytosis ability and tolerance induction potential. Thus, pediatric patients newly diagnosed with T1D, with established disease and control subjects were recruited. Firstly, DCs subsets from peripheral blood were found quantitatively altered during the first year of disease, but recovered in the second year of progression. Secondly, to study the tolerogenic functionality of DCs, liposomes with phosphatidylserine (PS) were designed to mimic apoptotic beta cells, which are able to induce tolerance, as previously demonstrated by our group in DCs from adult patients with T1D. In this study, monocyte-derived DCs from pediatric patients with T1D and control subjects were assessed in terms of PS-liposomes capture kinetics, and transcriptional and phenotypic changes. DCs from pediatric patients with T1D were found to phagocyte PS-liposomes more slowly and less efficiently than DCs from control subjects, inversely correlating with disease evolution. Nonetheless, the transcription of PS receptors and immunoregulatory genes, cytokine profile, and membrane expression of immunological markers in DCs was consistent with tolerogenic potential after PS-liposomes phagocytosis. In conclusion, T1D progression in childhood entails altered peripheral blood DCs subsets, as well as impaired DCs phagocytosis, although tolerance induction could still function optimally. Therefore, this study provides useful data for patient follow-up and stratification in immunotherapy clinical trials.
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Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/metabolismo , Suscetibilidade a Doenças , Tolerância Imunológica , Fagocitose/imunologia , Adolescente , Autoantígenos/imunologia , Autoimunidade , Biomarcadores , Plasticidade Celular/imunologia , Criança , Pré-Escolar , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunomodulação , Masculino , Fagocitose/genéticaRESUMO
BACKGROUND: The lack of continuity between health-related quality of life (HRQoL) instruments designed for children and adults hinders change analysis with a life course approach. To resolve this gap, EuroQol (EQ) developed the EQ-5D-Youth (EQ-5D-Y), derived from the EQ-5D for adults. Few studies have assessed the metric properties of EQ-5D-Y in children with specific chronic conditions, and none have done so for children with type I diabetes mellitus (T1DM). OBJECTIVE: This study aimed to evaluate the acceptability, validity, reliability, and responsiveness of the EQ-5D-Y in children and adolescents with T1DM, when administered online. METHODS: Participants with T1DM were consecutively recruited from July to December 2014, from a list of potential candidates aged 8-19 years, who attended outpatient pediatric endocrinology units. Before every quarterly routine visit, participants received an email/telephone reminder to complete the online version of two generic HRQoL questionnaires: EQ-5D-Y and KIDSCREEN-27. The EQ-5D-Y measures five dimensions, from which an equally weighted summary score was constructed (range: 0-100). Completion rate and distribution statistics were calculated. Construct validity was evaluated through known group comparisons based on general health, acute diabetic decompensations, mental health, family function, and a multitrait, multimethod matrix between EQ-5D-Y and KIDSCREEN by using Spearman correlations. Construct validity hypotheses were stated a priori. Reliability was assessed with the intraclass correlation coefficient and responsiveness by testing changes over time and calculating the effect size. Reliability and responsiveness were tested among the stable and improved subsamples defined by a KIDSCREEN-10 index change of <4.5 points or ≥4.5 points, respectively, from the first to the fourth visit. RESULTS: Of the 136 participants, 119 (87.5%) responded to the EQ-5D-Y at the last visit. The dimensions that showed higher percentages of participants with problems were "having pain/discomfort" (34.6%) and "worried/sad/unhappy" (28.7%). The mean (SD) of the EQ-5D-Y summary score was 8.5 (10.9), with ceiling and floor effects of 50.7% and 0%, respectively. Statistically significant HRQoL differences between groups defined by their general health (excellent/very good and good/regular/bad) and mental health (Strengths and Difficulties Questionnaire score ≤15 and >16, respectively) were found in three EQ-5D-Y dimensions ("doing usual activities," "having pain/discomfort," and "feeling worried/sad/unhappy"), summary score (effect size for general health and mental health groups=0.7 and 1.5, respectively), and KIDSCREEN-10 index (effect size for general health and mental health groups=0.6 and 0.9, respectively). Significant differences in the EQ-5D-Y dimensions were also found according to acute diabetic decompensations in "looking after myself" (P=.005) and according to family function in "having pain/discomfort" (P=.03). Results of the multitrait, multimethod matrix confirmed three of the four relationships hypothesized as substantial (0.21, 0.58, 0.50, and 0.46). The EQ-5D-Y summary score presented an intraclass correlation coefficient of 0.83. Statistically significant change between visits was observed in the improved subsample, with an effect size of 0.7 (P<.001). CONCLUSIONS: These results support the use of the EQ-5D-Y administered online as an acceptable, valid, reliable, and responsive instrument for evaluating HRQoL in children and adolescents with T1DM.
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Diabetes Mellitus Tipo 1/psicologia , Qualidade de Vida/psicologia , Adolescente , Adulto , Criança , Doença Crônica , Feminino , Humanos , Internet , Masculino , Inquéritos e Questionários , Adulto JovemRESUMO
Type 1 diabetes (T1D) is a chronic metabolic disease of unknown etiology that results from ß-cell destruction. The onset of the disease, which arises after a long asymptomatic period of autoimmune attack, may be followed by a relapsing and remitting progression, a phenomenon that is most evident during the partial remission phase (PR). This stage lasts for a few months, shows minor requirements of exogenous insulin and could be explained by a recovery of immunological tolerance. This study aims to identify new biomarkers at early stages of pediatric T1D that reflect immunoregulatory changes. To that end, pediatric patients with T1D (nâ¯=â¯52) and age-related control subjects (nâ¯=â¯30) were recruited. Immune response-related molecules and lymphocyte subsets were determined starting at T1D onset and until the second year of progression. Results showed that circulating TGF-ß levels decreased during PR, and that betatrophin concentration was increased in all the considered stages without differing among studied checkpoints. Moreover, an increase of regulatory T, B and NK subsets was found during T1D progression, probably reflecting an attempt to restore self-tolerance. By contrast, a reduction in monocyte levels was observed at the early stages of diabetes. The results reveal significant changes in immunological parameters during the different early stages of T1D in children, which could ultimately serve as potential biomarkers to characterize the progression of T1D.
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Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/sangue , Biomarcadores/sangue , Índice de Massa Corporal , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/sangue , Progressão da Doença , Feminino , Humanos , Memória Imunológica , Subpopulações de Linfócitos/metabolismo , Masculino , Monócitos/metabolismo , Hormônios Peptídicos/sangue , Projetos Piloto , Indução de Remissão , Fator de Crescimento Transformador beta/sangueRESUMO
BACKGROUND: vitamin D deficiency in children is still a global health problem. Measuring free 25-hydroxyvitamin D concentrations could provide a better estimate of the vitamin D status than total 25-hydroxyvitamin D (25(OH)D) levels. OBJECTIVE: To assess the relationship between measured free vitamin D (m-f25(OH)D) and calculated free 25(OH)D (c-f25(OH)D), total 25(OH)D, intact parathyroid hormone (iPTH) and other markers of phosphocalcic metabolism. To establish serum m-f25(OH)D concentrations corresponding to a total 25(OH)Dâ¯>â¯50â¯nmol/L which is accepted as vitamin D-sufficiency status in children. DESIGN: Prospective cohort study. SETTING: January and February 2017 in a Mediterranean population. PATIENTS: healthy children. MEASUREMENTS: m-f25(OH)D and vitamin D binding protein (VDBP) by ELISA. Free 25(OH)D was calculated using the formula described by Bikle. RESULTS: m-f25(OH)D directly correlated with total 25(OH)D (r:0.804,pâ¯<â¯.001), serum calcium (r:0.26,p:0.035), and c-f25(OH)D (r:0.553,p:0.016); and inversely with iPTH (r:-0.374, p:0.002), alkaline phosphatase (r:-0.28, p:0.026), and age (r:-0.289, p:0.018). Total 25(OH)D correlated with the same parameters as m-f25(OH)D except for serum calcium. However, c-f25(OH)D correlated only with total 25(OH)D and VDBP, both included in the calculation formula. Multiple regression analysis showed that m-f25(OH)D variations were independently explained by calcium (ß:0.156, p:0.026) and total 25(OH)D (ß:0.043, pâ¯<â¯.001). The optimal m-f25(OH)D cut-off for discriminating between insufficient and sufficient total 25(OH)D was >9.8â¯pmol/L (Area Under Curve (AUC): 0.897 (95% confidence interval (CI): (0.798-0.958); pâ¯<â¯.001; sensitivity:72.7% (95%CI: 49.8-89.3); specificity: 95.5% (95%CI: 84.5-99.4)). CONCLUSIONS: Directly measured free vitamin D correlated better with markers of phosphocalcic metabolism than total 25(OH)D and c-f25(OH)D in a population of healthy children.
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Doenças Assintomáticas , Calcifediol/sangue , Fenômenos Fisiológicos da Nutrição Infantil , Estado Nutricional , Deficiência de Vitamina D/sangue , Proteína de Ligação a Vitamina D/sangue , Adolescente , Biomarcadores/sangue , Calcifediol/química , Calcifediol/deficiência , Calcifediol/metabolismo , Cálcio/sangue , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Hospitais Universitários , Humanos , Masculino , Ambulatório Hospitalar , Hormônio Paratireóideo/sangue , Estudos Prospectivos , Valores de Referência , Sensibilidade e Especificidade , Solubilidade , Deficiência de Vitamina D/diagnóstico , Proteína de Ligação a Vitamina D/metabolismoRESUMO
RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.
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Dano ao DNA , DNA Fúngico/química , DNA Ribossômico/química , RNA Polimerase I/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Raios Ultravioleta , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
PURPOSE: To test whether the systematic monitoring of health-related quality of life (HRQOL) in clinical practice in Spanish pediatric patients with T1DM helps improve their daily life in a multicenter longitudinal study. METHODS: One hundred thirty-six patients participated, recruited from five centers in Barcelona, Spain (72 girls, mean age 13.4 years). Complete data were collected for 119 patients (85%). Pediatricians were randomly assigned to the HRQOL intervention (n = 70), or control group (n = 49). The intervention group discussed the results of HRQOL face to face with the physician, quarterly over a year. The control group received care as usual. HRQOL was assessed using KIDSCREEN-27 collected online. Standardized mean differences (effect size, ES) and generalized estimating equation (GEE) were computed to compare group differences between baseline and follow-up, taking into account sociodemographic and clinical variables. RESULTS: Statistically significant higher scores were seen in the intervention group at follow-up for the dimensions of Psychological well-being (ES = 0.56), School environment (ES = 0.56), and the KIDSCREEN-10 index (ES = 0.63). No differences were found in the control group. GEE analysis showed an improvement in HRQOL at follow-up with statistically significant association of the intervention on Psychological well-being (B = 4.32; p 0.03 for the interaction of group by follow-up) and School environment (B = 4.64; p 0.02 for the same interaction term). CONCLUSIONS: Routine assessment and face-to-face patient-physician discussion of HRQOL results improved HRQOL scores after a year of follow-up, especially in Psychological well-being and school environment. The results support the routinary use of HRQOL assessment in clinical practice.
Assuntos
Diabetes Mellitus Tipo 1/psicologia , Perfil de Impacto da Doença , Adolescente , Feminino , Humanos , Estudos Longitudinais , Masculino , Espanha , Inquéritos e QuestionáriosRESUMO
Type 1 diabetes (T1D) is a metabolic disease of unknown aetiology that results from the autoimmune destruction of the ß-cells. Clinical onset with classic hyperglycaemic symptoms occurs much more frequently in children and young adults, when less than 30% of ß-cells remain. Exogenous insulin administration is the only treatment for patients. However, due to glucose dysregulation, severe complications develop gradually. Recently, an increase in T1D incidence has been reported worldwide, especially in children. Shortly after diagnosis, T1D patients often experience partial remission called "honeymoon phase," which lasts a few months, with minor requirements of exogenous insulin. In this stage, the remaining ß-cells are still able to produce enough insulin to reduce the administration of exogenous insulin. A recovery of immunological tolerance to ß-cell autoantigens could explain the regeneration attempt in this remission phase. This mini-review focuses on the remission phase in childhood T1D. Understanding this period and finding those peripheral biomarkers that are signs of immunoregulation or islet regeneration could contribute to the identification of patients with a better glycaemic prognosis and a lower risk of secondary complications. This remission phase could be a good checkpoint for the administration of future immunotherapies.