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Browsing by Author "Carvalho, C."

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  • APOE isoforms in focal epilepsies: an association study in a Portuguese population
    Publication . Martins da Silva, A.; Leal, B.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Branco, R.C.; Ferreira, J.; Costa, P.; Martins da Silva, B.
    Purpose: Apolipoprotein E (ApoE) is the main lipoprotein secreted in brain. It has a critical immuno-modulatory function, influences neurotransmission and is involved in repairing damaged neurons. ApoE e4 allele is an isoform of ApoE with altered protein function previously associated with refractoriness and early onset epilepsy. Our purpose was to investigate if apoE isoforms are risk factors for partial epilepsy and to correlate genotypes with anti-epileptic drug response. Methods: A cohort of 230 epileptic patients with partial epilepsies from the outpatient clinic at HSA-CHP [109F, 121M; mean age = 44 13 years, age of onset = 15 13 years; 168 patients with Drug Refractory Epilepsy (DRE)] was compared with a cohort of 301 healthy individuals (HI) in a case control study. ApoE isoforms were genotype by RFLP-PCR methodology. Results: ApoE e4 allele frequency was higher in epileptic group when compared with HI (10.6% vs. 7.6%, p = n.s., OR = 1.44, 95% CI: 0.945–2.20). Anti-epileptic Drug response was not influenced by apoE isoforms. Conclusion: Our results suggest that ApoE e4 may be a risk factor for partial epilepsy development. ApoE e4 is associated with CNS network instability, with lower protection against oxidative and inflammatory cascade. These could influence neuronal growth and recovery leading to a chronic vicious cycle of damage and neuronal loss contributing to seizures development. These observations should be confirmed in a larger cohort.
    2013-06-17Conference object Restricted access Show more
  • Are Portuguese women of childbearing age exposed to environmental mercury? The One Health perspective
    Publication . Santiago, S; Namorado, S.; Dias, C.M.; Nascimento, A.; Martins, C.; Santos, M.; Carvalho, C.; Assunção, R.
    One health, a transdisciplinary approach, recognizes the interconnection between human, animals and their shared environment. Fish and seafood are important sources of high-biological value proteins, omega-3 fatty acids and essential minerals. However, it can contain environmental contaminants, such as mercury compounds. Long-lived predatory fish species, such as tuna or swordfish, are an important human exposure source. Thus, only through a transdisciplinary approach, namely using one health perspective, is it possible to properly tackle the issue of mercury at different levels. Methylmercury, the most toxic mercury form, mainly targets the central nervous system, and the prenatal period represents a period of greatest vulnerability regarding neurodevelopmental effects on the fetus. Portugal has a tradition of high consumption of fishery and aquaculture products, higher than in the European Union (EU) countries and above both EU and world averages. The present research aimed to evaluate the exposure of Portuguese women of childbearing age to mercury through human biomonitoring and to determine mercury contamination in fish available in Portuguese markets. For this study, 300 Portuguese women of childbearing age (25 to 44 years) were randomly selected in a cross-sectional epidemiological study carried out in Portugal (INSEF, http://www.insef.pt/) in 2015. Also, 24 different species of fish and fishery and aquaculture products acquired on the Portuguese market and representative of Portuguese consumption were selected. This study reinforces the need to develop and implement Portugal risk communication strategies focused on selecting fish species with lower mercury levels to protect susceptible populations from exposure to this chemical while simultaneously promoting the important health benefits of fish consumption and applying a One Health approach.
    2022-11-03Conference object Open access Show more
  • Association between vitamin D receptor (VDR) gene polymorphisms and systemic lupus erythematosus in Portuguese patients
    Publication . Carvalho, C.; Marinho, A.; Leal, B.; Bettencourt, A.; Boleixa, D.; Almeida, I.; Farinha, F.; Costa, P.P.; Vasconcelos, C.; Silva, B.M.
    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown origin, in which both genetic and environmental factors are involved. One such environmental factor is vitamin D, a vital hormone that plays a specific function in the immune system homeostasis, acting through a nuclear receptor (VDR) expressed in all immune cells. Several polymorphisms of the gene that encodes this receptor have been described. Though inconsistently, these polymorphisms have been associated with clinical manifestations and SLE development.The aim of this study was to determine the possible association between VDR gene polymorphisms (BsmI, ApaI, TaqI e FokI) and SLE susceptibility and severity, in a cohort of lupus patients from the north of Portugal.A total of 170 patients (F = 155, M = 15; age = 45 ± 13.4 years) with SLE (diagnosed according the American College of Rheumatology criteria) with at least five years of disease evolution and followed in the Autoimmune Disease Clinical Immunology Unit of Centro Hospitalar do Porto were studied. Patients and 192 ethnicity-matched controls were genotyped for BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236) and FokI (rs2228570) polymorphisms by TaqMan allelic discrimination assay. Disease severity was assessed by SLICC damage score, number of affected organs, number of severe flares and pharmacological history.SLE patients with the CT genotype of FokI polymorphism have a higher SLICC value (p = 0.031). The same result was observed for the group of patients with the TT genotype of TaqI polymorphism (p = 0.046). No differences were observed in VDR genotype between patients and controls. Also, we observed that the other clinical features analysed were not influenced by VDR polymorphisms.Our study confirms a possible role of VDR gene polymorphisms in SLE. A positive association was found between VDR polymorphisms and SLE severity (chronic damage). The presence of CT genotype of FokI and TT genotype of TaqI seems to confer a worse prognosis and may constitute a risk factor for higher long-term cumulative damage in SLE patients.
    2015-07Journal article Restricted access Show more
  • CCR5-Delta32: Implications in SLE development
    Publication . Carvalho, C.; Calvisi, S.L.; Leal, B.; Bettencourt, A.; Marinho, A.; Almeida, I.; Farinha, F.; Pinho-Costa, P.; Silva, B.M.; Vasconcelos, C.
    Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with strong genetic and environmental components. Previous studies have shown increased levels of several chemokines in active SLE. C-C chemokine receptor type 5 (CCR5) is involved in the recruitment of inflammatory cells into tissues, and mechanisms modulating CCR5 expression and function may interfere in SLE development, influencing the clinical course of the disease. The aim of this study was to evaluate the possible association between the CCR5{increment}32 base-pair deletion polymorphism and SLE disease in a group of Portuguese patients. A total of 219 patients with SLE and 205 healthy individuals were studied. The frequency of CCR5[del]32 heterozygotes was lower in patients with SLE than in controls (8% vs. 15% OR = 0.5162; P = 0.0319), suggesting a protective association between CCR5[del]32 allele and SLE. These results highlight the protective role of Th1 cells that express CCR5 in SLE pathogenesis.
    2013-10-29Journal article Restricted access Show more
  • Circulating microRNAs as potential biomarkers for genetic generalized epilepsies: a three microRNA panel
    Publication . Martins‐Ferreira, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Chorão, R.; Freitas, J.; Samões, R.; Boleixa, D.; Lopes, J.; Ramalheira, J.; Silva, B.M.; Martins da Silva, A.; Costa, P. P.; Leal, B.
    Background and purpose: Genetic generalized epilepsies (GGEs) encompass a group of syndromes of mainly genetic causes, characterized by the involvement of both hemispheres. MicroRNAs (miRNAs) are small non-coding RNAs with a critical role in the regulation of neuronal biological processes through gene expression modulation. Dysregulated miRNA expression has been shown in epilepsy. Due to their stability in biological fluids like serum, miRNAs have assumed a prominent role in biomarker research. Our aim was to evaluate circulating levels of three miRNAs in GGE patients and assess their putative diagnostic value. Methods: MiR-146a, miR-155 and miR-132 were quantified by real-time polymerase chain reaction in the serum of 79 GGE patients (47 women, 32 men, 35.1 ± 12.4 years) and 67 healthy individuals (41 women, 26 men, 42.4 ± 10.1 years). Relative expression values were calculated using the 2-ΔΔCt method. Receiver operating characteristic curve analysis was performed to assess diagnostic value. MiRNA expression was correlated with clinicopathological features. Results: Serum levels of miR-146a and miR-155 were significantly upregulated in GGE patients relative to controls (3.13 and 6.05, respectively). Combined miR-146a, miR-155 and miR-132 serum levels performed well as a diagnostic biomarker, discriminating GGE patients from controls with an area under the curve of 0.85, 80% specificity and 73% sensitivity. Conclusions: Our results indicate that miR-146a, miR-155 and miR-132 may partake in GGE epileptogenesis. A panel of three circulating miRNAs with potential value as a GGE biomarker is reported for the first time. Novel biomarkers may help to identify new treatment targets and contribute to improved patients' quality of life through earlier diagnosis and a more precise prognosis.
    2019-12-15Journal article Restricted access Show more
  • Compensatory T-Cell Regulation in Unaffected Relatives of SLE Patients, and Opposite IL-2/CD25-Mediated Effects Suggested by Coreferentiality Modeling
    Publication . Fesel, C.; Barreto, M.; Ferreira, R.C.; Costa, N.; Venda, L.L.; Pereira, C.; Carvalho, C.; Morães-Fontes, M.F.; Ferreira, C.M.; Vasconcelos, C.; Viana, J.F.; Santos, E.; Martins, B.; Demengeot, J.; Vicente, A.M.
    In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.
    2012-03-29Journal article Open access Show more
  • Expression of adenosine kinase in human mesial temporal lobe epilepsy with hippocampal sclerosis: A preliminary study
    Publication . Leal, B.; Rangel, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Zenatti, L.; Santos, A.; Magalhães, T.; Martins da Silva, A.; Correia de Sá, P.; Martins da Silva, B.; Costa, P.P.
    Background: Adenosine is a ubiquitous homeostatic molecule that acts as an “endogenous neuromodulator”. Adenosine attenuates neuronal activity either presynaptically by inhibiting neurotransmitter release or by controlling neurotransmitter responsiveness at post-synaptic sites. Unbalanced adenosine metabolism has been implicated in pathological conditions such as epilepsy. Adenosine kinase (ADK), synthetized by astrocytes, is the key regulator of extracellular adenosine levels in the brain. Evidences from experimental studies support a role for ADK in brain injury associated with astrogliosis, a morphological hallmark of Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS). In fact, expression of astrocytic ADK was found to be increased in the hippocampus and temporal cortex of MTLE-HS patients. Overexpression of ADK decreases extracellular adenosine and consequently may cause seizures. The aim of this study was to characterize ADK gene expression in MTLE-HS patients. Methods: Previous studies used immunohistochemistry and Western blot analysis to investigate ADK expression. Here we quantified the expression levels of ADK by Real-Time PCR in the hippocampus (lesional and peri-lesional cortical area) of 10 MTLE-HS patients submitted to surgery as compared with 9 autopsy controls with no history of neurological disorders. Results: Our results showed that ADK expression levels were similar in the hippocampus and temporal cortex of MTLE-HS patients when compared to healthy controls. Conclusion: Our preliminary data demonstrate that ADK expression levels are not altered in MTLE-HS. These results do not preclude post-transcriptional ADK abnormalities at both protein and functional levels. Our results should be confirmed in a larger cohort as well as with complementary methodologies.
    2013-09-22Conference object Restricted access Show more
  • Expression of miR146-a, an inflammation-associated microRNA, in Mesial Temporal Lobe Epilepsy
    Publication . Leal, B.; Carvalho, C.; Chaves, J.; Bettencourt, A.; Freitas, J.; Lopes, J.; Ramalheira, J.; Martins da Silva, A.; Costa, P. P.; Martins da Silva, B.
    Background: Neuroinflammation appears as an important epileptogenic mechanism. MicroRNAs (miRNA) are small non-coding RNA molecules that function as post-transcriptional regulators of gene expression. MicroRNas control different biological process including immune system homeostasis and function. Several evidences, both in patients and animal studies, have demonstrated an abnormal brain expression of miR-146a in Mesial Temporal Lobe Epilepsy. Knowing that miR expression is very stable in biological fluids such as plasma or serum our aim was to characterize miR146a expression in serum of MTLE patients. Methods: Expression levels of miR146a and U6B small nuclear RNA gene (reference gene) were quantified by Real-Time PCR in serum of 14 MTLE patients all with Hippocampal Sclerosis (6F, 8M, mean age= 44.1±11.7 years, age of onset= 13.5±10.6 years, 7 with Febrile Seizures antecedents). A group of 10 healthy individuals was used as control. Relative expression values were calculated using the 2-ΔΔCt method. Results: We observed that expression of miR146a was 2 fold higher in MTLE-HS patients than in controls. Conclusion: The results obtained in serum are in accordance with the results obtained from brain tissue of epileptic patients. This may confirm that miR-146a is a suitable biomarker of epileptogenesis. Additionally, it is thought that miR-146a has a role in fine-tuning the response to cytokines during epileptogenesis. Nevertheless its importance in epilepsy development it is yet not fully understood. The comprehension of this role may be relevant for the development of new therapeutic strategies.
    2015-05-20Conference object Open access Show more
  • How genetic characterization of Narcolepsy and hypersomnia is useful on phenotype definition
    Publication . Martins da Silva, A.; Lopes, J.; Ramalheira, J.; Carvalho, C.; Costa, P.P.; Silva, B.M.
    Introduction. The determination of HLA class II genotype is widely used to confirm the diagnosis of narcolepsy with or without cataplexy. It is known from studies carried out in the 80’s and 90’s that genetic markers, particularly HLA-DR2 (HLA-DRB1*15) and later DQB1*06:02, are strongly associated with susceptibility to narcolepsy (N). First studies in 1984 showed values of 100% of positive HLA-DR2 (Langdon et al; Lancet 1984) in narcoleptic patients with cataplexy (NC). Mignot et al (Sleep 1997) found values of 76.1% of HLA-DQB1*06:02 positive in NC and 40.9% in N. More recent studies emphasize difference between children and adults for HLA-DQB1*06:02. Values of 93.7% (adults) vs 92.6% (children) in NC and a frequency of 78,6% in adults vs 52.9% in children were found in N (Nevsimalova et al; J Neurol 2013). The dissemination of HLA genotyping was the result of two convergent reasons: i) the method is reliable, easy to perform and reassures the clinician; ii) the assay is less invasive than other methodologies, namely those involving cerebrospinal fluid (CSF) samples. Another contributing factor is the wide acceptance of the hypothesis of an autoimmune origin for Narcolepsy (a clinical field in which the relevance of HLA system is generally accepted). This hypothesis finds support in the virtually absent levels of hypocretin peptides in the CSF of patients with NC, which is postulated to be due to the autoimmune destruction of hypocretin producing neurons (Burgess et al; J Neurosci 2012). Aims. To evaluate the contribution of genetic markers (HLA) to the differential diagnosis between narcolepsy with (NC) or without cataplexy (N) and hypersomnia (H) and their relevance in the context of our population (Northern Portugal). Patients and methods. A cohort of 113 patients with sleep and hypersomnia complaints were observed at the Outpatient Sleep Clinic from Hospital Santo Antonio/CH Porto and were assessed by clinical, night sleep polygraphic recording, MSLT on the following day, blood sampling in a routine method. Data from laboratory parameters was confronted with the clinical diagnostic hypothesis. Clinical reevaluation of the patients was considered if results did not match. Of these patients, classified as NC, N or H (according to ICSD2, 2005), 38 were NC (age at testing: mean, 32.8 years; median, 30 years); 13 N (age at testing: mean, 34.2 years; median: 36 years); 62 patients had H (age at testing: mean, 38.2 years; median, 40 years). We used a control population (CP) of 206 reportedly healthy individuals from the same geographic origin. The allele frequencies of the control population were confirmed and compared with a larger cohort of another population (2500 individuals) from the central and south regions of the country. Results. The frequency of HLA-DQB1*06:02 allele was overrepresented in N and NC patients (46% and 71% respectively), as expected, and the p value is extremely significant for NC (p < 0.0000). HLA-DQB1*02 frequency was also increased in the population with H when compared with the CP (55% vs 34%; p = 0.00396). Interestingly the frequency of the HLADQB1*03 allele was decreased in the NC vs CP group (34% vs 56%; p =0.012153). No differences were found in the HLA-DQB1*06:03 frequency between the cohort of patients and the control population. Conclusions. The HLA-DQB1*06:02 allele, a susceptibility factor to other autoimmune disorders (e.g.: MS, SLE, sarcoidosis, sclerosing cholangitis), was confirmed as a susceptibility allele also to NC in our population. The frequency of this allele in our NC patients (71%) is within the range of other studies. This value is lower when compared to studies concerning only patients with severe cataplexy (frequencies between 85-95%). Some of the differences could be due to phenotypic uncertainty or to the clinical picture evolution in different age groups (Nevsimalova et al; J Neurol 2013). Also a modification of hypocretin levels, by circadian or other oscillations and the influence of environmental factors (infections, head trauma, immunization) can be involved. The role of the potential regeneration of CNS tissue is also a subject to be explored. Given these uncertainties, genetic characterization has the potential to enhance the ability to carry out differential diagnosis among diverse excessive daytime sleepiness phenotypes, helping in the distinction of diverse entities corresponding to fundamentally different biological processes. Finally a matter to be explored is the role of HLADQB1*06:03 allele, considered by some authors as a protective factor to NC (Hor et al; Nat Genet 2010) (Van der Heide et al; Sleep 2012). Our study did not confirm this assumption.
    2013-03-16Conference object Unknown Show more
  • Human herpes virus 6B and mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS): is there a link?
    Publication . Leal, B.; Castelo Branco, R.; Rangel, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Honavar, M.; Melo Pires, M.; Santos, A.; Magalhães, T.; Lopes, J.; Ramalheira, J.; Martins da Silva, A.; Costa, P.P.; Martins da Silva, B.
    Purpose: Human Herpesvirus 6 (HHV-6) is a ubiquitous virus acquired mainly during the first 2 years of life. Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) is the most frequent pharmacoresistant epilepsy. One of the most common antecedents of MTLE-HS is febrile seizures (FS). Although the aetiology of MTLE-HS remains unclear, evidences suggest that HHV-6 infection could be implicated. The objective of this study was to investigate the presence of HHV-6B DNA in the hippocampus and adjoining temporal cortex of MTLE-HS patients submitted to surgery. Methods: A total of 22 MTLE-HS (13 females and nine males) cases were studied. The mean age at surgery was 39 9 years and mean age at onset of seizures was 10 6 years. These study cohort was compared to a group of 10 epileptic patients without MTLE-HS (six females, four males; mean age = 26 15 years) and with autopsy material from nine individuals without neurological disease. HHV-6B DNA was identified by real-time PCR with specific TaqMan probes. Results: We detected HHV-6B DNA in only one hippocampus from a MTLE-HS patient. This patient had a disease duration of 36 years and a history of febrile seizures in childhood. None of the non-MTLE or controls specimens showed positivity for HHV-6B. Conclusions: Our findings do not support a relevant etiologic role for HHV-6B in MTLE-HS, at least in this population. However, the possible role of viral infection in MTLE-HS epileptogenic process, in individual cases, cannot be excluded.
    2013-06-17Conference object Restricted access Show more