Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors.

Disease is a neurodegenerative disorder characterised by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days' post-transplant. This long-lasting response was associated with Tumour necrosis factor alpha expression in microglial cells. In vitro, conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase-positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumour necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumour necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease.

Downregulation of Plasma Membrane Ca2+ ATPase driven by tyrosine hydroxylase-Gal4 reduces Drosophila lifespan independently of effects in neurons.

In Drosophila melanogaster, several Gal4 drivers are used to direct gene/RNAi expression to different dopaminergic neuronal clusters. We previously developed a fly model of Parkinson's disease, in which dopaminergic neurons had elevated cytosolic Ca2+ due to the expression of a Plasma Membrane Ca2+ ATPase (PMCA) RNAi under the thyroxine hydroxylase (TH)-Gal4 driver. Surprisingly, TH-Gal4>PMCARNAi flies died earlier compared to controls and showed swelling in the abdominal area. Flies expressing the PMCARNAi under other TH drivers also showed such swelling and shorter lifespan. Considering that TH-Gal4 is also expressed in the gut, we proposed to suppress the expression specifically in the nervous system, while maintaining the activation in the gut. Therefore, we expressed Gal80 under the direction of the panneuronal synaptobrevin (nSyb) promoter in the context of TH-Gal4. nSyb-Gal80; TH-Gal4>PMCARNAi flies showed the same reduction of survival as TH-Gal4>PMCARNAi flies, meaning that the phenotype of abdomen swelling and reduced survival could be due to the expression of the PMCARNAi in the gut. In perimortem stages TH-Gal4>PMCARNAi guts had alteration in the proventriculi and crops. The proventriculi appeared to lose cells and collapse on itself, and the crop increased its size several times with the appearance of cellular accumulations at its entrance. No altered expression or phenotype was observed in flies expressing PMCARNAi in the dopaminergic PAM cluster (PAM-Gal4>PMCARNAi). In this work we show the importance of checking the global expression of each promoter and the relevance of the inhibition of PMCA expression in the gut.

Understanding the role of the blood brain barrier and peripheral inflammation on behavior and pathology on ongoing confined cortical lesions.

BACKGROUND: Inflammation in the Central Nervous System (CNS) is associated with blood brain barrier (BBB) breakdown during the early stages of Multiple Sclerosis (MS), indicating a facilitated entry of waves of inflammatory cells from the circulation to the CNS. In the progressive forms of MS, as the lesion becomes chronic, the inflammation remains trapped within the CNS compartment forming the slow evolving lesion, characterized by low inflammation and microglia activation at the lesions edges. The chronic expression of interleukin 1ß (IL-1ß) in the cortex induces BBB breakdown, demyelination, neurodegeneration, microglial/macrophage activation and impaired cognitive performance. The latter can be improved, as long as the BBB recovers and the lesion presents low inflammation. Here, we study the effects of peripheral inflammation on cortical central lesions after the restoration of the BBB, in order to elucidate the role of the peripheral inflammation on these lesions with intact BBB, as it occurs in the progressive forms of MS. MATERIALS AND METHODS: Cortical lesions and peripheral inflammation were induced by the chronic expression of IL-1ß using an adenovector. We performed histological, immunohistochemistry on brain tissue and behavioural analyses. RESULTS: The effects of the chronic expression of IL-1ß in the cortex resolved within 56 days. However, peripheral and sustained inflammation re-opened the BBB, allowing the reappearance of the neuroinflammatory processes within the cortical lesions, increased demyelination and neurodegeneration, and an increase of the behavioral symptoms, such as cognitive impairment and anxiety-like symptoms. CONCLUSIONS: The early treatment of peripheral inflammatory processes should be considered in order to protect the brain from exacerbation of the ongoing neurodegenerative process.

A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels.

BACKGROUND: Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. METHODS: Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. RESULTS: Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. CONCLUSIONS: We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

Plasma membrane calcium ATPase downregulation in dopaminergic neurons alters cellular physiology and motor behaviour in Drosophila melanogaster.

The accumulation of Ca2+ and its subsequent increase in oxidative stress is proposed to be involved in selective dysfunctionality of dopaminergic neurons, the main cell type affected in Parkinson's disease. To test the in vivo impact of Ca2+ increment in dopaminergic neurons physiology, we downregulated the plasma membrane Ca2+ ATPase (PMCA), a pump that extrudes cytosolic Ca2+ , by expressing PMCARNAi in Drosophila melanogaster dopaminergic neurons. In these animals, we observed major locomotor alterations paralleled to higher cytosolic Ca2+ and increased levels of oxidative stress in mitochondria. Interestingly, although no overt degeneration of dopaminergic neurons was observed, evidences of neuronal dysfunctionality were detected such as increases in presynaptic vesicles in dopaminergic neurons and in the levels of dopamine in the brain, as well as presence of toxic effects when PMCA was downregulated in the eye. Moreover, reduced PMCA levels were found in a Drosophila model of Parkinson's disease, Parkin knock-out, expanding the functional relevance of PMCA reduction to other Parkinson's disease-related models. In all, we have generated a new model to study motor abnormalities caused by increments in Ca2+ that lead to augmented oxidative stress in a dopaminergic environment, added to a rise in synaptic vesicles and dopamine levels.

Environmental enrichment improves cognitive symptoms and pathological features in a focal model of cortical damage of multiple sclerosis.

Multiple Sclerosis (MS) is a neuroinflammatory disease affecting white and grey matter, it is characterized by demyelination, axonal degeneration along with loss of motor, sensitive and cognitive functions. MS is a heterogeneous disease that displays different clinical courses: relapsing/remitting MS (RRMS), and MS progressive forms: primary progressive (PPMS) and secondary progressive (SPMS). Cortical damage in the progressive MS forms has considerable clinical relevance due to its association with cognitive impairment and disability progression in patients. One treatment is available for the progressive forms of the disease, but none are specific for cognitive deficits. We developed an animal model that reflects most of the characteristics of the cortical damage, such as cortical neuroinflammation, demyelination, neurodegeneration and meningeal inflammation, which was associated with cognitive impairment. Cognitive rehabilitation, exercise and social support have begun to be evaluated in patients and animal models of neurodegenerative diseases. Environmental enrichment (EE) provides exercise as well as cognitive and social stimulation. EE has been demonstrated to exert positive effects on cognitive domains, such as learning and memory, and improving anxiety-like symptoms. We proposed to study the effect of EE on peripherally stimulated cortical lesion induced by the long term expression of interleukin IL-1ß (IL-1ß) in adult rats. Here, we demonstrated that EE: 1) reduces the peripheral inflammatory response to the stimulus, 2) ameliorates cognitive deficits and anxiety-like symptoms, 3) modulates neurodegeneration, demyelination and glial activation, 4) regulates neuroinflammation by reducing the expression of pro-inflammatory cytokines and enhancing the expression of anti-inflammatory ones. Our findings correlate with the fact that EE housing could be considered an effective non- pharmacological therapeutic agent that can synergistically aid in the rehabilitation of the disease.

Restless Legs Syndrome in Dialysis Patients: Does the Dialysis Modality Influence Its Occurrence and Severity?

BACKGROUND: Restless legs syndrome (RLS) is more prevalent in chronic kidney patients than in the general population, but it is often diagnosed late and its predictors are unknown. PURPOSE: To diagnose RLS in a group of chronic kidney patients on dialysis, determine its frequency and severity, compare the prevalence and severity of the condition among dialytic modalities, and identify possible predictive factors in this population. METHODS: An observational and cross-sectional study with 326 patients who had been on dialysis for more than 3 months, 241 on hemodialysis (HD) and 85 on automatic peritoneal dialysis (APD), using the criteria established by the International Study Group on RLS for the diagnosis and the RLS Rating Scale to determine its severity. RESULTS: RLS was diagnosed in 19.3% of the patients, 52.4% with severe or very severe forms. Patients with and without RLS did not differ in clinical and demographic characteristics and dialytic modality; however, patients on APD presented higher RLS severity compared to the HD group. CONCLUSIONS: RLS is frequent in dialysis patients and occurs predominantly in its most severe forms; the dialytic modality seems to have no influence on its occurrence; however, it is more severe in patients on APD.

A new focal model resembling features of cortical pathology of the progressive forms of multiple sclerosis: Influence of innate immunity.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of unknown aetiology that causes neurological disabilities in young adults. MS displays different clinical patterns, including recurrent episodes with remission periods ("relapsing-remitting MS" (RRMS)), which can progress over several years to a secondary progressive form (SPMS). However, 10% of patients display persistent progression at the onset of disease ("primary progressive MS" (PPMS)). Currently, no specific therapeutic agents are available for the progressive forms, mainly because the underlying pathogenic mechanisms are not clear and because no animal models have been specifically developed for these forms. The development of MS animal models is required to clarify the pathological mechanisms and to test novel therapeutic agents. In the present work, we overexpressed interleukin 1 beta (IL-1ß) in the cortex to develop an animal model reflecting the main pathological hallmarks of MS. The treated animals presented with neuroinflammation, demyelination, glial activation, and neurodegeneration along with cognitive symptoms and MRI images consistent with MS pathology. We also demonstrated the presence of meningeal inflammation close to cortical lesions, with characteristics similar to those described in MS patients. Systemic pro-inflammatory stimulation caused a flare-up of the cortical lesions and behavioural symptoms, including impairment of working memory and the appearance of anxiety-like symptoms. Our work demonstrated induced cortical lesions, reflecting the main histopathological hallmarks and cognitive impairments characterizing the cortical pathology described in MS patients with progressive forms of the disease.

A influência da aparência dentofacial sobre a atração interpessoal de crianças aos 5 e 10 anos de idade / The influence of appearance dentofacial on interpersonal attraction of children to 5 and 10 years of age

O objetivo desta pesquisa foi avaliar a influência da aparência dentofacial sobre a atração interpessoal entre escolares de 5 e 10 anos de idade no município de Patos/PB. A presença de más oclusões pode ser considerada um problema de saúde pública, apresentando alta prevalência e impacto social devido à interferência na qualidade de vida dos indivíduos. Imagens fotográficas de dois meninos e duas meninas, de 5 e 10 anos, foram modificadas através de computação gráfica, obtendo-se, para cada criança, um padrão facial harmonioso (face equilibrada) e três desarmoniosos, correspondentes às más oclusões de Classe II, III e da face longa. Cada criança da amostra, de forma individual, recebeu uma cartela contendo as montagens fotográficas de crianças na mesma faixa etária para que selecionasse as faces, segundo suas preferências e rejeições, abordando os aspetos de amizade, agressividade e beleza. Observou-se que em relação ao contexto da amizade e da beleza o maior índice de rejeição foi associado à Classe II. Crianças mais agressivas foram associadas à face longa. Os resultados obtidos foram estatisticamente significativos. Concluiu-se que as más oclusões mais acentuadas atuam como interferência na interação social de crianças e pré-adolescentes, haja visto que padrões faciais desarmônicos foram apontados com maior frequência a quesitos envolvendo rejeição. (AU)

The aim of this study was to evaluate the influence of dentofacial appearance on interpersonal attraction among children 5 to 10 years old. The presence of malocclusion can be considered a public health issue, with high prevalence and social impact due to interference in the quality of life of individuals. Photographic images of two boys and two girls, 5 and 10 years old, were modified through computer graphics, obtaining, for each child, a harmonious facial pattern (face balanced) and three inharmonious, corresponding to Class II malocclusions, Class III, and long face. Each child in the sample, individually, received a card with the photo montages of children in the same age group to evaluate the faces according to their preferences and rejections, addressing aspects of friendship, aggression and beauty. It was observed that in relation to the context of friendship and beauty the highest rate of rejection was associated with Class II. More aggressive children were associated with long face. The re-sults obtained were statistically significant. It was conclued that the sharpest malocclusions act as interference in the social interaction of children and pre-teens, since there disharmonious facial patterns were mentioned most often questions involving the rejection.(AU)

Cell therapy for Parkinson's disease: Functional role of the host immune response on survival and differentiation of dopaminergic neuroblasts.

Parkinson's disease (PD) is a neurodegenerative disorder, whose cardinal pathology is the loss of dopaminergic neurons in the substantia nigra. Current treatments for PD have side effects in the long term and do not halt disease progression or regenerate dopaminergic cell loss. Attempts to compensate neuronal cell loss by transplantation of dopamine-producing cells started more than 30 years ago, leading to several clinical trials. These trials showed safety and variable efficacy among patients. In addition to variability in efficacy, several patients developed graft-induced dyskinesia. Nevertheless, they have provided a proof of concept that motor symptoms could be improved by cell transplantation. Cell transplantation in the brain presents several immunological challenges. The adaptive immune response should be abolished to avoid graft rejection by the host. In addition, the innate immune response will always be present after transplanting cells into the brain. Remarkably, the innate immune response can have dramatic effects on the survival, differentiation and proliferation of the transplanted cells, but has been hardly investigated. In this review, we analyze data on the functional effects of signals from the innate immune system on dopaminergic differentiation, survival and proliferation. Then, we discussed efforts on cell transplantation in animal models and PD patients, highlighting the immune response and the immunomodulatory treatment strategies performed. The analysis of the available data lead us to conclude that the modulation of the innate immune response after transplantation can increase the success of future clinical trials in PD by enhancing cell differentiation and survival. This article is part of a Special Issue entitled SI: PSC and the brain.

Fibulin-2 is a key mediator of the pro-neurogenic effect of TGF-beta1 on adult neural stem cells.

Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.

Chronic systemic IL-1ß exacerbates central neuroinflammation independently of the blood-brain barrier integrity.

Peripheral circulating cytokines are involved in immune to brain communication and systemic inflammation is considered a risk factor for flaring up the symptoms in most neurodegenerative diseases. We induced both central inflammatory demyelinating lesion, and systemic inflammation with an interleukin-1ß expressing adenovector. The peripheral pro-inflammatory stimulus aggravated the ongoing central lesion independently of the blood-brain barrier (BBB) integrity. This model allows studying the role of specific molecules and cells (neutrophils) from the innate immune system, in the relationship between central and peripheral communication, and on relapsing episodes of demyelinating lesions, along with the role of BBB integrity.

In vivo biocompatibility versus degree of conversion of resin-reinforced cements in different time periods.

This study focused on test the null hypothesis that there is no difference between the degree of conversion and biocompatibility of different resin reinforced glass ionomer cements (RRGICs). Forty-eight male Wistar rats were used, distributed into four groups (n = 12), as follows: Group C (Control, polyethylene), Group FOB (Fuji Ortho Band), Group UBL (Ultra band Lok), and Group MCG (Multicure Glass), in subcutaneous tissue. The events of edema, necrosis, granulation tissue, multinuclear giant cells, young fibroblasts, and collagen formation were analyzed at 7, 15, and 30 days. The degree of conversion was evaluated by the Fourier method. Biocompatibility and degree of conversion were assessed using the Kruskal-Wallis and Dunn tests, and ANOVA and Tukey's test, respectively (P < 0.05). It was observed that, there was significant difference between Groups FOB and UBL for the presence of young fibroblasts at 15 days (P = 0.034) and between the Control and MCG Groups for the presence of multinucleated giant cells at 30 days (P = 0.009). Monomer conversion increased progressively until day 30, with significant difference between Group FOB and Groups UBL and MCG (P = 0.013) at 15 days. The null hypothesis was partially accepted, Fuji Ortho Band showed a less monomer conversion and a smaller number of young fibroblasts in the time of 15 days.

Isolated GH deficiency due to a GHRH receptor mutation causes hip joint problems and genu valgum, and reduces size but not density of trabecular and mixed bone.

CONTEXT: The GH/IGF-I axis is important for bone growth, but its effects on joint function are not completely understood. Adult-onset GH-deficient individuals have often reduced bone mineral density (BMD). However, there are limited data on BMD in adult patients with untreated congenital isolated GH-deficient (IGHD). We have shown that adult IGHD individuals from the Itabaianinha, homozygous for the c.57+1G>A GHRHR mutation, have reduced bone stiffness, but BMD and joint status in this cohort are unknown. OBJECTIVE: The goal is to study BMD, joint function, and osteoarthritis score in previously untreated IGHD adults harboring the c.57+1G>A GHRHR mutation. DESIGN: This is a cross-sectional study. METHODS: Areal BMD by dual-energy X-ray absorptiometry was measured in 25 IGHD and 23 controls (CO). Volumetric BMD (vBMD) was calculated at the lumbar spine and total hip. Joint function was assessed by goniometry of elbow, hips, and knees. X-rays were used to measure the anatomic axis of knee and the severity of osteoarthritis, using a classification for osteophytes (OP) and joint space narrowing (JSN). RESULTS: Genu valgum was more prevalent in IGHD than CO. The osteoarthritis knees OP score was similar in both groups, and knees JSN score showed a trend to be higher in IGHD. The hips OP score and JSN score were higher in IGHD. Areal BMD was lower in IGHD than CO, but vBMD was similar in the two groups. Range of motion was similar in elbow, knee, and hip in IGHD and CO. CONCLUSIONS: Untreated congenital IGHD due to a GHRHR mutation causes hip joint problems and genu valgum, without apparent clinical significance, reduces bone size, but does not reduce vBMD of the lumbar spine and hip.

Ortodontia e disfunção de ATM: revisão crítica / Orthodontics and ATM’s dysfunction: critical review

A articulação temporomandibular (ATM), por estar intimamente relacionada com os dentes, pode ser afetada por interferências traumáticas na oclusão. Uma série de fatores etiológicos tem sido descritos como potencialmente capazes de originar disfunção e, recentemente, dentre eles, coloca-se que o tratamento ortodôntico possa ser um deles. Devido à grande prevalência entre a população, tais desordens têm sido bastante pesquisadas e mantidas como questões complexas. Achou-se importante pesquisar o assunto através de uma revisão de literatura no intuito de compreender melhor a relação entre tratamento ortodôntico e desordem temporomandibular

The ATM, for being intimately related with the teeth, can be affected by traumatic interferences in the occlusion. There are many etiologic agents able to cause the dysfunction and recently, among them, is placed that the orthodontic treatment can be one these. Due to the great prevalence among the population, such disorders have been researched enough and maintained inside subjects. We thought important to research the subject through a revision of the literature in intention of understanding better the relationship between orthodontic treatment and DTM.