4-Phenylbutyrate Attenuates the ER Stress Response and Cyclic AMP Accumulation in DYT1 Dystonia Cell Models

Dystonia is a neurological disorder in which sustained muscle contractions induce twisting and repetitive movements or abnormal posturing. DYT1 early-onset primary dystonia is the most common form of hereditary dystonia and is caused by deletion of a glutamic acid residue (302/303) near the carboxyl-terminus of encoded torsinA. TorsinA is localized primarily within the contiguous lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), and is hypothesized to function as a molecular chaperone and an important regulator of the ER stress-signaling pathway, but how the mutation in torsinA causes disease remains unclear. Multiple lines of evidence suggest that the clinical symptoms of dystonia result from abnormalities in dopamine (DA) signaling, and possibly involving its down-stream effector adenylate cyclase that produces the second messenger cyclic adenosine-3′, 5′-monophosphate (cAMP). Here we find that mutation in torsinA induces ER stress, and inhibits the cyclic adenosine-3′, 5′-monophosphate (cAMP) response to the adenylate cyclase agonist forskolin. Both defective mechanins are corrected by the small molecule 4-phenylbutyrate (4-PBA) that alleviates ER stress. Our results link torsinA, the ER-stress-response, and cAMP-dependent signaling, and suggest 4-PBA could also be used in dystonia treatment. Other pharmacological agents known to modulate the cAMP cascade, and ER stress may also be therapeutic in dystonia patients and can be tested in the models described here, thus supplementing current efforts centered on the dopamine pathway

A produção do cuidado a usuários com hipertensão arterial e as tecnologias em saúde

Objetivou-se com este trabalho analisar a utilização da tecnologia das relações na produção do cuidado a usuários com hipertensão arterial. Trata-se de estudo qualitativo, realizado em oito Unidades de Saúde da Família de Jequié-BA. Os sujeitos do estudo foram formuladores da política, profissionais de saúde e usuários, totalizando dezesseis informantes. Em 2010, realizou-se entrevista semiestruturada, observação sistemática e análise documental. Para apreciação dos dados utilizou-se análise de conteúdo temática. Os resultados evidenciaram que os profissionais, sobretudo enfermeiros e agentes comunitários de saúde, utilizam tecnologias leves na busca da integralidade. Por outro lado, alguns profissionais demonstraram prática biologicista, com ênfase em normas e procedimentos. Conclui-se que há necessidade de mudança na interação entre trabalhador e usuário, considerando-se a singularidade e a autonomia do usuário e família na construção de novas formas de cuidado

Determinants of intensive insulin therapeutic regimens in patients with type 1 diabetes: data from a nationwide multicenter survey in Brazil

Background: To evaluate the determinants of intensive insulin regimens (ITs) in patients with type 1 diabetes (T1D).Methods: This multicenter study was conducted between December 2008 and December 2010 in 28 public clinics in 20 Brazilian cities. Data were obtained from 3,591 patients (56.0% female, 57.1% Caucasian). Insulin regimens were classified as follows: group 1, conventional therapy (CT) (intermediate human insulin, one to two injections daily); group 2 (three or more insulin injections of intermediate plus regular human insulin); group 3 (three or more insulin injections of intermediate human insulin plus short-acting insulin analogues); group 4, basal-bolus (one or two insulin injections of long-acting plus short-acting insulin analogues or regular insulin); and group 5, basal-bolus with continuous subcutaneous insulin infusion (CSII). Groups 2 to 5 were considered IT groups.Results: We obtained complete data from 2,961 patients. Combined intermediate plus regular human insulin was the most used therapeutic regimen. CSII was used by 37 (1.2%) patients and IT by 2,669 (90.2%) patients. More patients on IT performed self-monitoring of blood glucose and were treated at the tertiary care level compared to CT patients (p < 0.001). the majority of patients from all groups had HbA1c levels above the target. Overweight or obesity was not associated with insulin regimen. Logistic regression analysis showed that economic status, age, ethnicity, and level of care were associated with IT (p < 0.001).Conclusions: Given the prevalence of intensive treatment for T1D in Brazil, more effective therapeutic strategies are needed for long term-health benefits.Farmanguinhos/Fundacao Oswaldo Cruz/National Health MinistryBrazilian Diabetes SocietyFundacao do Amparo a Pesquisa do Estado do Rio de JaneiroConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Estado Rio de Janeiro, Unit Diabet, BR-20551030 Rio de Janeiro, BrazilBaurus Diabet Assoc, São Paulo, BrazilFed Univ São Paulo State, Diabet Unit, São Paulo, BrazilFed Univ Hosp Porto Alegre, Porto Alegre, BrazilUniv Hosp São Paulo, Diabet Unit, São Paulo, BrazilUniv Fed Rio de Janeiro, Rio de Janeiro, BrazilUniv Fed Ceara, Fortaleza, Ceara, BrazilSanta Casa Misericordia, Belo Horizonte, MG, BrazilSanta Casa Misericordia São Paulo, São Paulo, BrazilUniv Fed Amazonas, Manaus, Amazonas, BrazilHosp Geral de Bonsucesso, Rio de Janeiro, BrazilHosp Univ Clementino Fraga Filho IPPMG, Rio de Janeiro, BrazilUniv Hosp São Paulo, São Paulo, BrazilFac Ciencias Med Santa Casa São Paulo, São Paulo, BrazilUniv São Paulo, Inst Crianca, Hosp Clin, São Paulo, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Hosp Clin, Ribeirao Preto, BrazilAmbulatorio Fac Estadual Med Sao Jose Rio Preto, Ribeirao Preto, BrazilEscola Paulista Med, Ctr Diabet, Ribeirao Preto, BrazilClin Endocrinol Santa Casa Belo Horizonte, Belo Horizonte, MG, BrazilUniv Estadual Londrina, Londrina, BrazilUniv Fed Parana, Hosp Clin, Porto Alegre, RS, BrazilInst Crianca Com Diabet Rio Grande Sul, Rio Grande Do Sul, RS, BrazilGrp Hosp Conceicao, Inst Crianca Com Diabet, Porto Alegre, RS, BrazilHosp Univ Santa Catarina, Florianopolis, SC, BrazilInst Diabet Endocrinol Joinville, Joinville, BrazilHosp Reg Taguatinga, Brasilia, DF, BrazilHosp Geral Goiania, Goiania, Go, BrazilCtr Diabet & Endocrinol Estado Bahia, Goiania, Go, BrazilUniv Fed Maranhao, Sao Luis, BrazilCtr Integrado Diabet & Hipertensao Ceara, Fortaleza, Ceara, BrazilUniv Fed Sergipe, Aracaju, BrazilHosp Univ Alcides Carneiro, Campina Grande, BrazilHosp Univ Joao de Barros Barreto, Belem, Para, BrazilFed Univ São Paulo State, Diabet Unit, São Paulo, BrazilUniv Hosp São Paulo, Diabet Unit, São Paulo, BrazilUniv Hosp São Paulo, São Paulo, BrazilEscola Paulista Med, Ctr Diabet, Ribeirao Preto, BrazilWeb of Scienc

Regional differences in clinical care among patients with type 1 diabetes in Brazil: Brazilian Type 1 Diabetes Study Group

Background\ud To determine the characteristics of clinical care offered to type 1 diabetic patients across the four distinct regions of Brazil, with geographic and contrasting socioeconomic differences. Glycemic control, prevalence of cardiovascular risk factors, screening for chronic complications and the frequency that the recommended treatment goals were met using the American Diabetes Association guidelines were evaluated.\ud \ud Methods\ud This was a cross-sectional, multicenter study conducted from December 2008 to December 2010 in 28 secondary and tertiary care public clinics in 20 Brazilian cities in north/northeast, mid-west, southeast and south regions. The data were obtained from 3,591 patients (56.0% females and 57.1% Caucasians) aged 21.2 ± 11.7 years with a disease duration of 9.6 ± 8.1 years (<1 to 50 years).\ud \ud Results\ud Overall, 18.4% patients had HbA1c levels <7.0%, and 47.5% patients had HbA1c levels ≥ 9%. HbA1c levels were associated with lower economic status, female gender, age and the daily frequency of self-blood glucose monitoring (SBGM) but not with insulin regimen and geographic region. Hypertension was more frequent in the mid-west (32%) and north/northeast (25%) than in the southeast (19%) and south (17%) regions (p<0.001). More patients from the southeast region achieved LDL cholesterol goals and were treated with statins (p<0.001). Fewer patients from the north/northeast and mid-west regions were screened for retinopathy and nephropathy, compared with patients from the south and southeast. Patients from the south/southeast regions had more intensive insulin regimens than patients from the north/northeast and mid-west regions (p<0.001). The most common insulin therapy combination was intermediate-acting with regular human insulin, mainly in the north/northeast region (p<0.001). The combination of insulin glargine with lispro and glulisine was more frequently used in the mid-west region (p<0.001). Patients from the north/northeast region were younger, non-Caucasian, from lower economic status, used less continuous subcutaneous insulin infusion, performed less SBGM and were less overweight/obese (p<0.001).\ud \ud Conclusions\ud A majority of patients, mainly in the north/northeast and mid-west regions, did not meet metabolic control goals and were not screened for diabetes-related chronic complications. These results should guide governmental health policy decisions, specific to each geographic region, to improve diabetes care and decrease the negative impact diabetes has on the public health system.We thank Mrs. Karianne Aroeira Davidson, Mrs. Anna Maria Ferreira, Mrs. Elisangela Santos and Sandro Sperandei for their technical assistance.This work was supported by grants from Farmanguinhos/Fundação Oswaldo Cruz/National Health Ministry, the Brazilian Diabetes Society, Fundação do Amparo à Pesquisa do Estado do Rio de Janeiro, and Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia

Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environmen

Model of torsinA modulation of cAMP.

<p>In healthy fibroblast cells (left panel), forskolin stimulates adenylate cyclase (AC) to synthesis cAMP, and there is no presence of Endoplasmic Reticulum (ER) stress in normal conditions. In absence of torsinA or in presence of mutant torsinA, torsinAΔE (right panel), cells present increased ER stress and lower cAMP accumulation. However, 4-PBA treatment rescues both ER stress and cAMP accumulation defects in DYT1 dystonia cell models by unoknow mechanism (s).</p

4-PBA alleviates ER stress in dystonia models.

<p>(A) Healthy control fibroblast cells lines (average of 3 lines: HF6, HF19 and GM02912) and DYT1 patient fibroblast lines (average of three lines: GM02306, GM02551 and GM03211) were treated for 18 hours in the absence or presence of 2.5 or 5 mM concentrations of 4-PBA, then challenged or not with 1 µM thapsigargin (Thaps.) for 3 hours. We have not observed significant differences in sliced XBP1 (sXBP1) levels, an ER stress marker, upon 4-PBA treatments (2.5 and 5 mM) in healthy control or DYT1 fibroblast cells. (B) As shown in (A), healthy fibroblast cells lines (3 lines represented in white 1–3: HF6, HF19, and GM02912) and DYT1 patient fibroblast cell lines (3 lines represented in black 4–6: GM02306, GM02551, and GM03211) were treated for 18 hours in the absence or presence of 10 mM concentration of 4-PBA, then challenged or not with 1 µM thapsigargin (Thaps.) for 3 hours. DYT1 fibroblasts cell lines have increased levels of mRNA expression levels of sliced XBP1 (sXBP1), an ER stress marker, compared to healthy controls (**P<0.01; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments) when stimulated with thapsigargin. However, when DYT1 fibroblast cells were pre-treated with 10 mM 4-PBA, they showed reduced sXBP1 levels, similar to healthy control lines. A 1.2 reduction in the sXBP levels was observed when DYT1 fibroblast cells challenged with thapsigargin, and treated with 4-PBA (**P<0.01; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments). The results indicate that the presence of torsinAΔE induces ER stress in fibroblast cells, however, 10 mM 4-PBA treatment decreases ER stress in DYT1 patient lines.</p

cAMP defects in dystonia models are rescued by 4-PBA treatments.

<p>(A) Healthy control fibroblast cells (3 lines represented in white 1–3: HF6, GM01651, and GM02912) and DYT1 patient fibroblast cells (3 lines represented in black 4–5: GM02306, GM02551, and GM03211) were pre-treated with or without 4-PBA for 18 hours, followed by 10 µM forskolin stimulation for 30 minutes. As observed previously, DYT1 fibroblast lines have significantly lower levels of cAMP accumulation after forskolin stimulation compared to healthy control fibroblast lines (**P<0.01; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments). However, the 4-PBA treatments also significantly rescued cAMP accumulation deficiency in DYT1 patient fibroblast lines comparable to control fibroblasts cAMP levels (*P<0.5; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments). (B) No significant changes were observed in the intracellular ATP levels from healthy control cells (HF6, GM01651, GM02912) and DYT1 fibroblast cells (GM2306, GM02551, GM3211).</p

Absence of torsinA or presence of torsinAΔE, downregulates cAMP production in dystonia animal models.

<p>(A) Lysates from mouse embryonic fibroblasts (MEFs) cells from controls (torsinA^+/+) and MEFs from knockout torsinA mice (torsinA^-/-) were resolved by SDS-PAGE, and immunoblotted with antibodies specific to mouse torsinA (torsinA), and β-Actin, showing the absence of torsinA in torsinA^-/- MEFs. (B) To analyze differences in cAMP levels in torsinA^+/+, and torsinA^-/- MEFs, the cAMP production was measured under basal conditions and in response to the direct adenylate cyclase agonist forskolin (10 µM). Although no significant differences in basal cAMP production were observed in MEFs cells, there was a 5-fold reduction (***P<0.001; Student t-test, n = 3 experiments) in torsinA^-/- MEFs after forskolin stimulation compared to control torsinA^+/+ MEFs. (C) Primary mixed cortical and striatal neuronal cultures of individual E15 embryos from controls littermates (torsinA^+/+), heterozigous Knockout torsinA (torsinA^+/-), and homozigous Knockout torsinA (torsinA^-/-). No signicant differences in basal cAMP production were observed, however a 1.5 fold reduction of cAMP levels was also observed in torsinA^-/- neurons (**P<0.01; Student t-test, n = 3 experiments) after forskolin stimulation compared to control torsinA^+/+, neurons. (D) Same results were found in knock-in dystonia models. A 1.2 fold reduction in the cAMP production was obseved in heterozygotes Knock-in (torsinA^WT/ΔE) neurons (*P<0.05; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments), and a 1.4 fold reduction in homozygotes Knock-in (torsinA^ΔE/ΔE) neurons (*P<0.05; one-way ANOVA and Tukey's post hoc comparison, n = 3 experiments) in forskolin-stimulated cAMP production cells compared to control torsinA^wt/wt neurons. (D) Since adenylate cyclase catalyzes the conversion of ATP to cAMP, intracellular ATP levels were measured from torsinA^-/- MEFs and control torsinA^+/+ MEFs (E), and in heterozygous or homozygous Knock-in neurons (F), when compared to controls. The intracellular ATP levels were determined from cell lysates using the ATP-Glo™ Bioluminometric Cell Viability Assay. No significant differences were found in the ATP levels (n = 3 experiments).</p