The relationship between inflammatory markers and hippocampal and extra-hippocampal atrophy patterns in patients with temporal lobe epilepsy

Orientadores: Fernando Cendes, Ana Carolina CoanTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Introdução: A epilepsia atinge 1 a 2 % da população mundial, sendo a epilepsia de lobo temporal associada a esclerose hipocampal (ELT-EH) a forma mais frequente em adultos. Estudos de imagem já determinaram que a ELT-EH apresenta redução volumétrica cerebral difusa, não sendo restrita à região do hipocampo. Há evidências de que a inflamação tem um importante papel na neuroexcitabilidade e que alterações na regulação inflamatória podem gerar degeneração neuronal e induzir crises convulsivas Objetivos: Definir a associação de marcadores inflamatórios séricos e epilepsias, além de características clínicas, de EEG e padrões de alteração de neuroimagem em pacientes com ELT. Materias e Métodos: Foram incluídos no estudo 490 pacientes com diagnóstico clínico e eletroencefalográfico de epilepsia e um grupo de 166 controles sem doenças neurológicas. Os pacientes foram divididos entre os com ELT (246) e pacientes com outras epilepsias. Os indivíduos foram submetidos a coleta de sangue para a avaliação dos marcadores inflamatórios e 86 desses realizaram ressonância magnética (RM) de crânio. O volume de estruturas subcorticais e espessura de regiões corticais foram analisadas pelo programa FreeSurfer. Os marcadores inflamatórios (IL-1, IL-2, IL-4, IL-6, IL-10, IL-17, TNF? e seus receptores sTNFr1 e sTNFr2, BDNF, CTNF, IFN?, NGF, GDNF) foram analisados de maneira cega aos dados clínicos pelas técnicas Enzyme-Linked Immunosorbent Assay (ELISA) e Cytometric Bead Array (CBA). Resultados: Os fatores inflamatórios não estão correlacionados com a idade e gênero dos indivíduos e nem com o tempo de doença dos pacientes (r>0,3; p>0,05). Os níveis séricos de BDNF, NGF, sTNFr2 e a NT3 foram elevados enquanto que os níveis de TNF, sTNFr1, IFN? e das interleucinas foram reduzidos quando avaliamos os pacientes, em geral ou os grupos ELT e outras epilepsias em relação aos controles. Mesmo padrão foi observado quando avaliados apenas os pacientes com malformação do desenvolvimento cortical, exceto o sTNR1 e IL10 que não foram significativos nessa última análise. O CNTF, NT4/5 e GDNF não foram diferentes entre pacientes e controles. O sTNFr2 se demonstrou um bom marcador para diferenciar pacientes e controles (curva ROC com AUC de 0,858). Analisando apenas os pacientes com ELT, O GDNF foi maior nos pacientes com pouca atividade epileptiforme ao EEG. O IL2 e o IL4 foram elevados nos pacientes com maior frequência de crises. Em relação à imagem, o TNF? sérico apresentou correlação inversa ao volume do tálamo ipsilateral. Discussão: Nosso estudo é um dos primeiros a avaliar uma extensa coorte de pacientes e realizar uma avaliação exploratória sobre a relação de marcadores inflamatórios, dados clínicos e de neuroimagem. Os marcadores inflamatórios apresentam diferentes papéis no sistema nervoso central e sua medição sérica nos ajuda a compreender melhor o papel desses nas epilepsias. Conclusão: Os marcadores inflamatórios estão claramente envolvidos na epilepsia e na perda neuronal, porém são necessários estudos específicos para compreender se estes são epifenômenos ou consequências das crisesAbstract: Introduction: Epilepsy affects 1% to 2% of the world population and temporal lobe epilepsy associated with hippocampal sclerosis (TLE-HS) is the most common epilepsy in adults. Imaging studies have already demonstrated that TLE patients present diffuse gray and white matter atrophy, not restricted to hippocampal region. Inflammation perform an important role in neuroexcitability. Changes on inflammatory regulation can lead to neuronal degeneration and induce seizures. Objectives: To define the association of serum inflammatory markers and epilepsies, as well as clinical characteristics, EEG and neuroimaging patterns in patients with TLE. Subjects and Methods: The study included 490 patients with clinical and electroencephalographic (EEG) diagnosis of epilepsy and a group with 166 controls without neurological diseases. The patients were classified as TLE-HS (246) and other epilepsies. All participants were invited to perform blood sampling (blood serum for inflammatory markers) and 86 performed magnetic resonance imaging (MRI). MRI cortical thickness and subcortical structures were analyzed with FreeSurfer software. The blood serum analysis of IL-1, IL-2, IL-4, IL-6, IL-10, IL-17, TNF? ,sTNFr1, sTNFr2, BDNF, CTNF, IFN?, NGF, GDNF was performed by Enzyme-Linked Immunosorbent Assay (ELISA) and Cytometric Bead Array (CBA). Results: The inflammatory markers did not present correlation with age and gender, or with epilepsy duration. The blood serum levels of BDNF, NGF, sTNFr2 e a NT3 were higher while TNF, sTNFr1, IFN? and interleukines were reduced in patients with epilepsy, TLE-HS and other epilepsies when compared with controls. The same pattern was observed in patients with malformation of cortical development, except for sTNFr1 and IL10. The CTNF, NT4/5 and GDNF were not different between patients and controls in any group. In ROC analysis the sTNFr2 was a good marker to separate patients from controls (AUC =0,858). Considering just patients, the GDNF presented higher serum levels in patients with less interictal epileptiform activity at EEG; and blood serum IL2 and IL4 were higher in patients with more frequent seizures. TNF? presented an inverse correlation with ipsilateral thalamus volume. Discussion: Our study is one of the first to evaluate an extensive cohort of patients and perform an exploratory assessment on the relationship between inflammatory markers, clinical data and imaging. Inflammatory markers play different roles in central nervous system and their serum measurement helps in better comprehending their relationship with epilepsy. Conclusion: The inflammatory markers are associated with epilepsy, but more specific studies are necessary to determine if they are epiphenomena or consequence of seizuresDoutoradoFisiopatologia MédicaDoutora em Ciências2015/17066-0;FAPESPCAPE

Food handling in the domestic environment: an online questionnaire study with respondents from 24 of 26 Brazilian states

Este estudo teve como objetivo avaliar o perfil das práticas de manipulação de alimentos no ambiente domiciliar no Brasil utilizando um questionário online. Um questionário contendo perguntas sobre comportamento doméstico em nível de higiene e manipulação de alimentos foi construído e disponibilizado por redes sociais. O questionário continha informações sobre o perfil dos participantes, suas práticas de pré preparo, preparo e pós-preparo de alimentos e a ocorrência de doenças transmitidas por alimentos (DTA). Obteve-se 701 respostas, os entrevistados foram 78,31% do sexo feminino e 21,68% do sexo masculino, com média de idade de 31,2 anos. A maioria (94,3%) possuia ensino superior completo ou incompleto. Na etapa de pré-preparo, os participantes avaliam o prazo de validade (97,28%) e a temperatura de armazenamento (44,79%) dos produtos no momento da compra. Em relação às práticas de manipulação dos alimentos, apenas alguns participantes lavavam as embalagens dos alimentos antes de armazená-los (31,95%) ou retiravam adornos ao lavar os alimentos (61,48%). A maioria dos participantes lavam as mãos (91,58%) e os vegetais (99,28%); entretanto, um grupo de entrevistados relatou lavar carne crua (27,81%) antes de prepará-la. Superfícies de corte como tábuas de plástico (50,36%) e de vidro (49,36%) foram os mais prevalentes no estudo. A maioria dos entrevistados não sabe há quanto tempo usa as tábuas de corte (67,62%) e utilizam a mesma superfície para manusear produtos crus e prontos para o consumo (84,17%). Quanto ao preparo, a maioria dos entrevistados declarou não verificar a temperatura dos alimentos durante o preparo (86,31%), ignorando a temperatura ideal de cozimento (88,26%). Em relação à ocorrência de DVA, 79,17% dos entrevistados relataram que já apresentaram sinais clínicos suspeitos associados a alimentos contaminados e 65,59% não procuraram atendimento médico. Nesse sentido, os participantes demonstraram desconhecimento sobre as práticas adequadas para a segurança dos alimentos no ambiente domiciliar, evidenciando a necessidade de realização de programas de educação em saúde com a população brasileira.Using an online questionnaire, this study evaluated the profile of a Brazilian population’s food handling practices in the home environment. The questionnaire, containing questions about domestic behavior in terms of hygiene and food handling, was built and available through social media sites. Information about the participants’ profiles, their food pre-preparation, food preparation, and food post-preparation practices, and the occurrence of foodborne diseases (FBDs) was included in the questionnaire. A total of 701 responses were obtained. The interviewees included 78.31% female participants and 21.68% male participants, with an average age of 31.2 years. Nearly all (94.3%) had a complete or incomplete higher education. In the pre preparation stage, the participants evaluated the shelf life (97.28%) and storage temperature (44.79%) of the products while purchasing them. Regarding food handling practices, only a few participants washed the food packages before storing them (31.95%) or removed hand jewelry or other adornments when washing food (61.48%). Most participants washed their hands (91.58%) and washed vegetables (99.28%). But a group of interviewees reported washing raw meat (27.81%) before preparing it. Cutting surfaces such as plastic (50.36%) and glass (49.36%) tops were the most prevalent in the study. Most respondents did not know how long they had been using their cutting boards (67.62%) and mentioned using the same surface to handle both raw and ready-to-eat products (84.17%). As for the preparation, most interviewees declared they did not check the food temperature during preparation (86.31%), ignoring the ideal cooking temperature (88.26%). Regarding the occurrence of FBDs, 79.17% of the interviewees reported having suspicious clinical signs associated with contaminated foods and 65.59% did not seek medical help. Thus, the participants demonstrated ignorance about adequate practices for food safety in the home environment, highlighting the need to conduct health education programs within the Brazilian population

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

HMGB1: A Common Biomarker and Potential Target for TBI, Neuroinflammation, Epilepsy, and Cognitive Dysfunction

High mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein released by glia and neurons upon inflammasome activation and activates receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR) 4 on the target cells. HMGB1/TLR4 axis is a key initiator of neuroinflammation. In recent days, more attention has been paid to HMGB1 due to its contribution in traumatic brain injury (TBI), neuroinflammatory conditions, epileptogenesis, and cognitive impairments and has emerged as a novel target for those conditions. Nevertheless, HMGB1 has not been portrayed as a common prognostic biomarker for these HMGB1 mediated pathologies. The current review discusses the contribution of HMGB1/TLR4/RAGE signaling in several brain injury, neuroinflammation mediated disorders, epileptogenesis and cognitive dysfunctions and in the light of available evidence, argued the possibilities of HMGB1 as a common viable biomarker of the above mentioned neurological dysfunctions. Furthermore, the review also addresses the result of preclinical studies focused on HMGB1 targeted therapy by the HMGB1 antagonist in several ranges of HMGB1 mediated conditions and noted an encouraging result. These findings suggest HMGB1 as a potential candidate to be a common biomarker of TBI, neuroinflammation, epileptogenesis, and cognitive dysfunctions which can be used for early prediction and progression of those neurological diseases. Future study should explore toward the translational implication of HMGB1 which can open the windows of opportunities for the development of innovative therapeutics that could prevent several associated HMGB1 mediated pathologies discussed herein

Characterization of genome-wide p53-binding sites upon stress response

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg2+. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (Kobs = 1.2 (±0.3) × 105 M−1) is tighter than that of MSK (2.9 (±1.0) × 104 M−1) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53+/+ cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53

miR-22 represses cancer progression by inducing cellular senescence

The microRNA miR-22 targets CDK6, SIRT1, and Sp1—genes involved in regulation of the senescence program—to suppress cell growth and proliferation

NAD(P)H Quinone Oxidoreductase Protects TAp63γ from Proteasomal Degradation and Regulates TAp63γ-Dependent Growth Arrest

BACKGROUND: p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative DeltaNp63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The DeltaNp63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63gamma, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. PRINCIPAL FINDINGS: We show that TAp63gamma can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63gamma, stabilizes it and protects it from this degradation. NQO1-mediated TAp63gamma stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63gamma-dependant p21 upregulation and TAp63gamma-induced growth arrest stimulated by doxorubicin. CONCLUSIONS/SIGNIFICANCE: Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63gamma. These findings appoint a direct role for NQO1 in the regulation of TAp63gamma expression, especially following stress and may therefore have clinical implications for tumor development and therapy

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements†

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein–DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process