Neuropsychiatric Features of a Cohort of Patients with Systemic Lupus Erythematosus

In order to establish if neuropsychiatric systemic lupus erythematosus (NPSLE) can be identified by any characteristic other than those used to diagnose the neuropsychiatric (NP) disease itself, we retrospectively reviewed 98 systemic lupus erythematosus (SLE) patients followed over a mean period of 10 years. NPSLE was identified in 22 patients. Stroke and generalized seizures were the most frequent NP manifestations. The NPSLE and non-NPSLE groups were similar with regard to demographic characteristics, ACR criteria, serum autoantibodies, and frequency of hypertension and hypercholesterolemia. Of note, compared to the non-NPSLE group, NPSLE was associated with a higher frequency of smoking (78 versus 26%), organ damage (73 versus 34%), and cumulative mortality rate (14 versus 7%). The series of patients was further analysed according to the presence of antiphospholipid syndrome (APS). Significantly, the interval between the onset of NP disease and SLE diagnosis was shorter in the APS(-) (0.3 ± 1 years) than in the APS(+) (5 ± 7 years) groups. Recurrence and/or persistence of NP events were only documented in the APS(-) group. Overall cumulative mortality was highest in NPSLE and in APS(+) patients with inadequate anticoagulation control, identifying an aspect that requires improved vigilance and the development of novel therapeutic modalities.

Juvenile Systemic Lupus Erythematosus in Portugal: Clinical and Immunological Patterns of Disease Expression in a Cohort of 56 Patients

Objective: To define the pattern of disease expression and to gain better understanding in patients with juvenile onset systemic lupus erythematosus (SLE) in Portugal. Methods: The features of unselected patients with systemic lupus erythematosus who had disease onset before the age of 18 years were retrospectively analysed in three Portuguese centres with Pediatric Rheumatology Clinic over a 24-year period (1987-2011). Demographic, clinical and laboratory manifestations, therapy and outcome were assessed. Results: A cohort of 56 patients with a mean age at disease onset of 12.6±4.04 years (mean±1SD) (range, 1.0-17.0 years) and a mean period of follow-up of 5.5±5.4 years. Forty six (82.1%) patients were female. The most common disease manifestations were musculoskeletal (87.5%), mucocutaneous (80.3%) and haematological abnormalities (75%). Lupus nephritis was diagnosed in 46.4% of patients and consisted of glomerular ne - phritis in all cases. Neuropsychiatric manifestations occurred in 21.4% but severe central nervous system complications were uncommon, as brain infarcts and organic brain syndrome in 4 (7.1%) patients. Antinuclear antibodies and anti-double stranded DNA were positive in most patients in (98.2% and 71.4% respectively), as well as low C3 and/or C4 were observed frequently (85.7%). Generally, most patients had a good response to therapy as demonstrated by a significant decreasing of SLEDAI score from disease presentation to the last evaluation. The SLEDAI at diagnosis, the maximum SLEDAI and the incidence of complications were significantly higher in patients with neurolupus and/or lupus nephritis. Therapy included oral steroids (87.5%), hydroxychloroquine (85.7%), azathioprine (55.4%), IV cyclophosphamide (28.6%) along with other drugs. Six (10.7%) patients were treated with rituximab. Long-term remission was achieved in 32%, disease was active in 68%, adverse reactions to therapy occurred in 53.6% and complications/severe manifestations in 23.2%. Two patients died, being active disease and severe infection the causes of death. Conclusions: This study suggests that in our patients the clinical and laboratory features observed were similar to juvenile systemic lupus erythematosus patients from other series. Clinical outcome was favourable in the present study. Complications from therapy were frequent. Objective: To define the pattern of disease expression and to gain better understanding in patients with juvenile onset systemic lupus erythematosus (SLE) in Portugal. Methods: The features of unselected patients with systemic lupus erythematosus who had disease onset before the age of 18 years were retrospectively analysed in three Portuguese centres with Pediatric Rheumatology Clinic over a 24-year period (1987-2011). Demographic,clinical and laboratory manifestations, therapy and outcome were assessed. Results: A cohort of 56 patients with a mean age at disease onset of 12.6±4.04 years (mean±1SD) (range, 1.0-17.0 years) and a mean period of follow-up of 5.5±5.4 years. Forty six (82.1%) patients were female. The most common disease manifestations were musculoskeletal (87.5%), mucocutaneous (80.3%) and haematological abnormalities (75%). Lupus nephritis was diagnosed in 46.4% of patients and consisted of glomerular ne - phritis in all cases. Neuropsychiatric manifestations occurred in 21.4% but severe central nervous system complications were uncommon, as brain infarcts and organic brain syndrome in 4 (7.1%) patients. Antinuclear antibodies and anti-double stranded DNA were positive in most patients in (98.2% and 71.4% respectively), as well as low C3 and/or C4 were observed frequently (85.7%). Generally, most patients had a good response to therapy as demonstrated by a significant decreasing of SLEDAI score from disease presentation to the last evaluation. The SLEDAI at diagnosis, the maximum SLEDAI and the incidence of complications were significantly higher in patients with neurolupus and/or lupus nephritis. Therapy included oral steroids (87.5%), hydroxychloroquine (85.7%), azathioprine (55.4%), IV cyclophosphamide (28.6%) along with other drugs. Six (10.7%) patients were treated with rituximab. Long-term remission was achieved in 32%, disease was active in 68%, adverse reactions to therapy occurred in 53.6% and complications/severe manifestations in 23.2%. Two patients died, being active disease and severe infection the causes of death. Conclusions: This study suggests that in our patients the clinical and laboratory features observed were similar to juvenile systemic lupus erythematosus patients from other series. Clinical outcome was favourable in the present study. Complications from therapy were frequent.

Neuropsychiatric Questionnaires in Systemic Lupus Erythematosus

Patients with systemic lupus erythematosus (SLE) can be affected by a multitude of neurologic and psychiatric symptoms with a wide range of prevalence and severity. Irrespectively from attribution to SLE or other causes, neuropsychiatric (NP) symptoms strongly impact short-term and long-term outcomes,thus NP evaluation during routine clinical practice in SLE should be undertaken regularly. The assessment of NP involvement in SLE patients is challenging and the available diagnostic tools fail to guarantee optimal diagnostic accuracy, sensitivity to changes as well as feasibility in routine clinical care. Standardised questionnaires (both physician-administered and self-reported) can offer valuable help to the treating physician to capture all possible NP syndromes; few SLE-specific NP questionnaire have been developed but validation in large cohort or cross-cultural adaptations are still pending. On the other hand, general instruments have been largely applied to SLE patients. Both kinds of questionnaires can address all possible NP manifestations either globally or, more frequently, focus on specific NP symptoms. These latter have been mainly used in SLE to detect and classify mild and subtle symptoms, more likely to be overlooked during routine clinical assessment such as headache, cognitive impairment and psychiatric manifestations. In conclusion, this literature review highlights a clear case for validation studies in this area and the wider implementation of questionnaires to assess NP involvement is still warranted. The broader use of such instruments could have important consequences; first of all, by standardising symptom assessment, a better definition of the prevalence of NP manifestation across different centres could be achieved. Secondly, prospective studies could allow for the evaluation of clinical significance of mild symptoms and their impact on the patient’s function and quality of life.

Study on solubility of pharmaceutical compounds in ionic liquids

Dissertation to obtain the degree of master in Chemical and Biochemical Engineering

Atherogenesis and atherosclerosis in primary antiphospholipid syndrome

ABSTRACT: In the late seventies the term “Haematological Stress Syndrome” defined some haematological abnormalities appearing in the course of acute and chronic disorders, such as raised plasma levels of fibrinogen (FNG) and factor VIII, reduced fibrinolytic activity and hyperviscosity. In the early nineties the “Membrane stress syndrome hypothesis” proposed the unification of the concepts of haematological stress syndrome with those of oxidation, inflammation and immune activation to explain the pathogenesis of the antiphospholipid syndrome (APS) Antiphospholipid antibodies, coagulation, fibrinolysis and thrombosis. This chapter investigated the occurrence of the “Haematological Stress Syndrome” and thrombosis in 144 participants positive for aPL detected by clotting and immune tests. Among the clotting assays for the detection of lupus anticoagulant, dilute Russell's viper venom time better correlated with a history of venous thrombosis than activated partial thromboplastin time (p<0.0002 vs p<0.009) and was the only test correlated with a history of arterial thrombosis (p<0.01). By regression analysis, serum levels of IgG anticardiolipin antibodies (aCL) associated with the number of venous occlusions (p<0.001). With regards to FNG and von Willebrand factor (vWF), the former rose by 36% (95% CI; 21%, 53%) and the latter by 50% (95% CI; 29%, 75%) at the first venous occlusion and remained unchanged after subsequent occlusions. At variance FNG rose by 45% (95% CI; 31%, 60%) per arterial occlusion and vWF by 27% (95% CI; 10%, 47%) per arterial occlusion throughout. The coagulation/fibrinolytic balance was cross-sectionally evaluated on 18 thrombotic PAPS patients, 18 subjects with persistence of idiopathic aPL and in healthy controls. Markers of thrombin generation prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and of fibrin turnover D-Dimer (D-D) were higher in thrombotic (p=0.006)and non-thrombotic subjects (p=0.0001) than in controls as were those of D-D (p<0.0001 and p=0.003 respectively). TAT levels did not differ. Gender analysed data revealed blunted tPA release (hence a negative venous occlusion test) in thrombotic females but neither in thrombotic males (p=0.01) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p<0.0002) and control females (p<0.02). The activity of factor XIII (FXIIIa) was evaluated was evaluated in 29 patients with PAPS, 14 persistent carriers of aPL without thrombosis, 24 thrombotic patients with inherited thrombophilia, 28 healthy controls and 32 patients with mitral and aortic valve prosthesis as controls for FXIII only. FXIIIa was highest in PAPS (p=0.001), particularly in patients with multiple (n=12) than single occlusion (p=0.02) and in correlation with PAI (p=0.003) and FNG (p=0.005). Moreover FXIIIa was strongly associated with IgG aCL and IgG anti-2GPI (p=0.005 for both) in the PAPS group and to a lesser degree in the aPL group (FXIIIa with IgG aCL, p=0.02, with IgG anti-2GPI, p=0.04). Altogether these results indicate: 1) a differential relationship of aPL, vWF and FNG with venous and arterial thrombosis; 2) heightened thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs; 3) enhanced FXIIIa that may contribute to atherothrombosis via increased fibrin/fibrinogen cross-linking. Lipid profile, lipid peroxidation and anti-lipoprotein antibodies in thrombotic primary antiphospholipid syndrome. Given the atherogenic lipid profile of SLE, the same possibility was explored in PAPS by comparing high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein AI (ApoAI), apolipoprotein B (ApoB), triglycerides (TG), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-2GPI) and C-reactive protein (CRP) in 34 thrombotic PAPS patients compared to 36 thrombotic patients with inherited thrombophilia (IT), to 18 subjects persistently positive for antiphospholipid antibodies (aPL) with no underlying autoimmune or non-autoimmune disorders and to 28 healthy controls. Average concentrations of HDL (p<0.0001), LDL (p<0.0001), CHO (p=0.0002), ApoAI (p=0.002) were lower in PAPS whereas average TRY was higher (p=0.01) than other groups. Moreover PAPS showed higher IgG anti-HDL (p=0.01) and IgG anti-ApoAI (p<0.0001) as well as greater average oxLDL-2GPI (p=0.001) and CRP (p=0.003). Within PAPS, IgG anti-HDL correlated negatively to HDL (p=0.004) and was an independent predictor of oxLDL-2GPI (p=0.009). HDL and ApoAI correlated negatively with CRP (p=0.001 and p=0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favouring low-grade inflammation and enhanced oxidation in thrombotic PAPS. Indeed plasma 8-epi-prostaglandin F2α (a very specific marker of lipid peroxidation) was significantly higher in 10 patients with PAPS than 10 age and sex matched healthy subjects (p=0.0002) and strongly related to the titre of plasma IgG aCL (r=0.89, p=0.0004). Hence oxidative stress, a major player in atherogenesis, also characterises PAPS. Nitric oxide and nitrative stress in thrombotic primary antiphosholipid syndrome. Oxidative stress goes hand in hand with nitrative stress and to address the latter plasma nitrotyrosine (NT, marker of nitrative stress), nitrite (NO2-) and nitrate (NO3-) were measured in 46 thrombotic PAPS patients, 21 asymptomatic but persistent carriers of antiphospholipid antibodies (PCaPL), 38 patients with inherited thrombophilia (IT), 33 patients with systemic lupus erythematosus (SLE) and 29 healthy controls (CTR). Average crude NT was higher in PAPS and SLE (p=0.01) whereas average plasma NO2- was lower in PAPS and average NO3- highest in SLE (p<0.0001). In PAPS, IgG aCL titer and number of vascular occlusions negatively predicted NO2-, (p=0.03 and p=0.001, respectively) whereas arterial occlusions and smoking positively predicted NO3- (p=0.05 and p=0.005). Moreover CRP (an inflammatory marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade inflammation and both phenomena may have implications for thrombosis and atherosclerosis in PAPS Inflammation and immune activation in thrombotic primary antiphospholipid syndrome. To investigate inflammation and immune activation in thrombotic PAPS high-sensitivity CRP (hs-CRP), serum amyloid A (SAA), oxLDL-2GPI, CRP bound to oxLDL-2GPI (CRP-oxLDL-2GPI) (as inflammatory markers) neopterin (NPT) and soluble CD14 (sCD14) (as immune activation markers) were measured by ELISA in 41 PAPS patients, in 44 patients with inherited thrombophilia (IT) and 39 controls (CTR). Compared to other groups, PAPS presented with higher plasma concentrations of inflammatory, hs-CRP (p=0.0004), SAA (p<0.01), CRP-oxLDL-2GPI (p=0.0004) and immune activation markers, NPT (p<0.0001) and sCD14 (p=0.007). By regression analysis SAA independently predicted thrombosis number (p=0.003) and NPT independently predicted thrombosis type (arterial, p=0.03) and number (p=0.04). These data confirm that low-grade inflammation and immune activation occur and relate to vascular features of PAPS. Antiphosholipid antibodies, haemostatic variables and atherosclerosis in thrombotic primary antiphospholipid syndrome To evaluate whether IgG aCL titre, haemostatic variables and the lipid profile bore any relationship to the intima media thickness (IMT) of carotid arteries high-resolution sonography was applied to the common carotid (CC), carotid bifurcation (CB) and internal carotid (IC) of 42 aPL subjects, 29 with primary thrombotic antiphospholipid syndrome and 13 with persistence of aPL in the absence of any underlying disorder. The following were measured: plasma FNG, vWF, PAI, homocysteine (HC), CHO, TG, HDL, LDL, platelet numbers and aCL of IgG and IgM isotype. By multiple regression analysis, IgG aCL titre independently predicted IMT at all carotid segments examined (p always <0.005). Plasma FNG and HC independently predicted IMT at the CB (p=0.001 and p<0.0001, respectively) and IC (p=0.03 and p<0.0001, respectively). These data strongly support an atherogenic role for IgG aCL in patients with aPL in addition to traditional risk factors. The atherosclerosis hypothesis was investigated in an age and sex-matched case-double-control study including 49 thrombotic PAPS patients (18 M, 31 F, mean age 37 ± 11), 49 thrombotic patients for IT and 49 healthy subjects. Average IMT was always greater in PAPS than control patients (CC: p=0.004, CB: p=0.013, IC: p=0.001). By dividing participants into age tertiles the IMT was greater in the second (CC: p=0.003, CB: p=0.023, IC: p=0.003) and third tertiles (CC: p=0.03, CB: p=0.004, IC: p=0.007). Conclusion: Coagulation activation, fibrinolysis depression, hightened fibrin turnover, oxidative and nitrative stress in parallel with low grade inflammation and immune activation characterise thrombotic PAPS: all these are early atherogenic processes and contribute to the demonstrated premature atherosclerosis that should be considered a clinical feature of PAPS.

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Dissertação para obtenção do Grau de Doutor em Engenharia Informática

Humoral response towards high density lipoprotein : a new mechanism for atherogenesis

RESUMO:Aterosclerose é uma das principais causas de morbilidade e mortalidade no mundo ocidental. É responsável, direta ou indiretamente, pela maior percentagem de gastos com a saúde na maioria dos países europeus. A “teoria lipídica” da aterosclerose, que se baseia na dislipidemia como causa primária para a doença vascular tem algumas implicações práticas importantes: permite a definição de linhas de orientação e protocolos simples e ainda estabelece alvos terapêuticos que podem ser atingidos na maior parte dos casos com a atual intervenção farmacológica. A associação da aterosclerose com o sistema imunológico (a “teoria imunológica”), forneceu por sua vez novas formas de explorar os mecanismos envolvidos e abriu novas perspetivas para um conhecimento mais completo da doença. No entanto, levanta dificuldades evidentes no que diz respeito às possibilidades terapêuticas. De todos os intervenientes no processo aterosclerótico (bioquímicos, imunológicos e anatómicos), as lipoproteínas de elevada densidade (HDL) são atualmente reconhecidas como um dos fatores mais importantes na aterogénese. Isto é baseado no reconhecimento das múltiplas propriedades anti-aterogénicas das HDL como por exemplo: a anti-oxidante, a anti-inflamatória e a antitrombótica, bem como o seu importante papel na melhoraria da função endotelial. Atualmente, é consensual que as funções anti-aterogénicas das HDL vão além do seu papel no transporte reverso do colesterol (RCT) e a importância das HDL no processo aterosclerótico baseia-se não apenas no seu papel protetor impedindo a formação da placa de ateroma, mas também na estabilização destas, prevenindo a sua ruptura e, consequentemente o evento trombótico. Como fundamentais no processo aterosclerótico estão reconhecidos dois principais conjuntos de eventos: um caracterizado por alterações no metabolismo das lipoproteínas que resultam em lipoproteínas pró-inflamatórias e pró-oxidantes que interagem com os componentes celulares da parede arterial e que conduzem à formação da placa de ateroma; o outro evento é a resposta imunológica desencadeada contra um novo conjunto de antigénios que por sua vez leva à produção de citoquinas pró-inflamatórias. Dada a complexidade da HDL e das suas múltiplas funções estas lipoproteínas tornaram-se um potencial alvo para a resposta auto-imune, e cujas consequências podem explicar algumas das associações identificados em estudos clínicos e epidemiológicos. Contudo esta interação entre o sistema imunológico e HDL nunca foi exaustivamente estudada. Portanto, pomos a hipótese de que em condições oxidativas e pró-inflamatórias, um aumento do antigénio (HDL) conduz a um consequente acréscimo na produção de anticorpos anti-HDL (aHDL) responsáveis pela alteração quantitativa e / ou qualitativa das HDL. O conceito de que estes anticorpos podem contribuir tanto para a evolução a longo prazo do processo aterosclerótico, como para o desencadeamento de eventos clínicos pode também explicar a heterogeneidade encontrada em cada doente e nos grandes estudos clínicos, no que diz respeito aos fatores de risco e outcomes clínicos. Para além disso, a confirmação desta hipótese pode permitir explicar porque é que as intervenções terapêuticas atualmente em desenvolvimento para aumentar os níveis de HDL, não conseguem mostrar a tão esperada redução do risco vascular. O objetivo geral desta tese foi identificar e caracterizar a resposta humoral contra os componentes da HDL, e avaliar possíveis mecanismos que possam contribuir para a modificação das propriedades anti-aterogénicas das HDL. Para alcançar este objetivo investigou-se: 1) A presença de anticorpos aHDL em doentes com lúpus eritematoso sistémico (SLE) e em doentes com manifestações clínicas de aterosclerose, como os doentes com doença arterial coronária (CAD), acidente vascular cerebral isquémico (IS) e diabetes tipo 2; 2) Os principais alvos antigénicos dentro do complexo das HDL e a associação entre os títulos de anticorpos aHDL e diferentes características clínicas destas doenças; 3) As modificações das funções normais associadas às HDL, em particular da função anti-oxidante e anti-inflamatória; 4) A atividade biológica dos anticorpos aHDL isolados do soro de doentes através de um conjunto de experiências in vitro de inibição da atividade da paraoxonase 1 (PON1) e da expressão de moléculas de adesão em culturas de células endoteliais. Para tal foi necessário estabelecer um método de isolamento dos anticorpos. Os anticorpos aHDL isolados do soro de doentes foram utilizados de forma a identificar as potenciais alterações dos sistemas celulares utilizados; 5) O efeito de fármacos usados no tratamento das dislipidemias, em particular o ácido nicotínico e as estatinas, na variação dos títulos de anticorpos aHDL através de ensaios clínicos randomizados, controlados com placebo e em dupla ocultação. Os métodos utilizados neste trabalho incluíram: técnicas imunológicas (como por exemplo, enzyme-linked immunoabsorbent assay - ELISA, ensaio imunoturbidimetrico e cromatografia de imuno-afinidade) técnicas bioquímicas (tais como a quantificação de atividade enzimática por espectrofotometria e por luminescência), experiências com cultura de células e citometria de fluxo. Os nossos resultados mostram que: 1) A presença de anticorpos aHDL, e mais especificamente anticorpos contra alguns do seus principais componentes como a apolipoproteína A-I (ApoA-I, principal apolipoproteína presente nas HDL) e a PON1 (o enzima que mais contribui para a propriedade anti-oxidante das HDL), quer em doentes com doenças auto-imunes, como o SLE, quer em doentes com manifestações clínicas de aterosclerose, como CAD, IS e diabetes tipo 2. Os doentes apresentaram títulos de anticorpos IgG aHDL, aApoA-I e aPON1 significativamente mais elevados do que controlos saudáveis com a mesma idade e sexo. 2) A correlação positiva estatisticamente significativa entre os títulos de aHDL e aApoA-I e aPON1 sugere que estes sejam dois dos principais alvos antigénicos dentro do complexo das HDL. Os anticorpos encontrados nestes doentes estão associados com a diminuição da atividade da PON1 e a uma redução da capacidade anti-oxidante total (TAC) do soro, um aumento dos biomarcadores de disfunção endotelial (como por exemplo dos metabolitos do óxido nítrico - NO2- e NO3-, as moléculas de adesão vascular e intracelular - VCAM-1 e ICAM-1 e os níveis de 3-nitrotirosina). Nos doentes com SLE os títulos destes estão associados a um aumento do dano cardiovascular e à atividade global da doença avaliados pelas escalas SLICC/ACR DI e BILAG score, respetivamente. Enquanto que nos doentes com diabetes tipo 2 estes anticorpos estão associados com um aumento dos níveis de glicemia em jejum (FGP) e hemoglobina glicada (HbA1c). 3) Após se ter estabelecido um método de isolamento dos anticorpos que permite isolar quantidades significativas de anticorpos do soro de doentes sem perder a sua especificidade, foi identificada a capacidade dos anticorpos isolados do soro de doentes inibirem de uma forma dependente da concentração a atividade da PON1 até um máximo de 70% no caso dos doentes com SLE e ente 7-52% no caso dos anticorpos isolados de doentes com CAD e IS. 4) O efeito anti-inflamatório das HDL na inibição da produção de VCAM-1 induzida por citoquinas (como o TNF-) foi revertido em mais de 80% pelos anticorpos aHDL isolados do soro de doentes. 5) A angiogenesis induzida por HDL através do aumento do fator de crescimento do endotélio vascular (VEGF) foi anulada em 65% pelos anticorpos aHDL isolados do soro de doentes. 6) Os atuais agentes farmacológicos disponíveis para aumentar as concentrações de HDL-C estão associados a um aumento dos títulos de anticorpos.-------- ABSTRACTAtherosclerosis is the major cause of morbidity and mortality in the western world. It is also responsible, directly or indirectly, for the highest percentage of health costs in most European countries. Despite the use of new technologies for the diagnosis of vascular disease and regardless of the major advances in treatment, the atherosclerosis-related clinical burden is still raising. The “lipid theory” of atherogenesis, which identifies dyslipidemia as the primary cause of this vascular disease has some important practical implications: it allows the definition of simple guidelines and establishes therapeutic targets which can be generally met with current pharmacologic intervention. The association between atherosclerosis an the immune system (the immune concept) has in turn provided new ways of exploring the mechanisms involved in this condition and has opened new perspectives in the understanding of the disease. However, it raises obvious difficulties when it comes to treatment options. Of all the players (biochemical, immunological and anatomical) involved in this matter, high-density lipoproteins (HDL) are currently recognised as one of the most important factors in atherogenesis. This is based on the recognition of HDL's multiple anti-atherogenic properties: anti-oxidant, anti-inflammatory and antithrombotic, as well as its capacity to improve endothelial function. Nowadays, it is widely recognized that the anti-atherogenic functions of HDL go beyond reverse cholesterol transport (RCT), and the importance of HDL is based not just on its ability to reduce atheroma formation but also on its ability to stabilise plaques, therefore preventing their rupture and ultimately thrombosis. Two main set of events have been recognised as fundamental in atherogenesis: one, characterized by lipoprotein metabolism alterations, resulting in pro-inflammatory and pro-oxidative lipoproteins, which interact with the normal cellular elements of the arterial wall leading to atheroma formation; the other, the immune cellular response towards new sets of antigens which lead to the production of pro-inflammatory cytokines. Given to HDL complexity and multiple functions this lipoprotein has became a potential target for an auto-immune response, the consequences of which may explain some of the association identified in epidemiological and clinical studies, though the interaction between the immune system and HDL has never been thoroughly addressed. Therefore, we hypothesized that under oxidative and pro-inflammatory conditions, the increase in the antigen (HDL) would lead to a consequent increase in the production of anti-HDL (aHDL) antibodies be responsible for quantitative and/or qualitative changes of HDL. The concept that these antibodies may contribute either to the long-term evolution of atherosclerosis or to the triggering of clinical events may also explain the heterogeneity found in individual patients and in large cohorts regarding risk factors and clinical outcomes. Moreover this may be a major breakthrough in understanding why therapeutic interventions that increase HDL levels, failed to show the anticipated reduction in vascular risk. The overall aims of this thesis were to identified and characterize the humoral response towards HDL components and to evaluate the possible mechanisms that may contribute to the modifications of the anti-atherogenic properties of HDL. To achieve this objective we investigated: 1) the presence of aHDL antibodies in patients with systemic lupus erythematosus (SLE) and in patients with atherosclerosis-related clinical events, such as coronary artery disease (CAD), ischemic stroke (IS) and type 2 diabetes; 2) the association between the titres of aHDL antibodies and different clinical features of these diseases; 3) the modifications of the anti-atherogenic properties of HDL; 4) the biologic effect of aHDL antibodies isolated from serum of patients on the anti-oxidant and anti-inflammatory properties of HDL; 5) the effect of different pharmacologic treatments for dyslipidemia on the prevalence and activity of aHDL antibodies. The methodologies used in this work included immunologic-related techniques (e.g. enzyme-linked immunoabsorbent assay – ELISA, immunoturbidimetric immunoassay and immunoaffinity chromatography), biochemical techniques (enzymatic assays with quantification by spectrophotometry and luminescence methods), cell culture experiments and flow cytometry. Our results indicate that: 1) The titres of IgG aHDL, anti-apolipoprotein A-I (aApoA-I) and anti-paraoxonase 1 (aPON1) antibodies were higher in patients with SLE, CAD, IS and type 2 diabetes when compared with age and sex matched healthy controls. 2) The antibodies found in these patients were associated with decreased PON1 activity, (the enzyme responsible for most of the anti-oxidant effect of HDL), reduced total anti-oxidant capacity (TAC) of serum and increased biomarkers of endothelial dysfunction (nitric oxide metabolites, adhesion molecules, nitrotyrosine). In patients with SLE the antibody titres were associated with an increase in disease-related cardiovascular damage and activity whereas in patients with type 2 diabetes they were directly related with the fasting glucose plasma (FGP) levels and the glycosylated haemoglobin (HbA1c). 3) The antibodies isolated from serum of our patients, directly inhibited HDL-associated PON1 activity in a dose dependent way ranging from 7 to 52%. 4) The anti-inflammatory effect of HDL, measured by the percentage of inhibition of the cytokine-induced production of vascular adhesion molecules (VCAM-1), was reduced in more than 80% by aHDL antibodies isolated from our patients. 5) The HDL-induced angiogenesis by increasing vascular endothelial growth factor (VEGF) levels was abrogated in 65% by the antibodies isolated from serum of patients. 6) The current available pharmacologic agents for increasing HDL-C concentrations were associated with an increase in the titres of IgG aApoA-I antibodies. This increase was higher in the extended release niacin when compared to statins probably due to their dampening effect on oxidative stress. In conclusion, aHDL antibodies are present in different pathologic conditions. aHDL antibodies represent a family of self-reacting immunoglobulins, of which ApoA-I and PON1 might be the most relevant targets. These antibodies are biologically active, interfering with the HDL anti-oxidant and anti-inflammatory properties and, consequently, with the atherosclerotic process. The pathogenic potential of these antibodies may lead to the identification of a new biomarker for vascular disease, whilst presenting itself as a novel target for a different treatment approach which may redefine the treatment strategies and clinical trials design for HDL interventions in the future.

Qualidade de vida relacionada com a saúde em doentes com lúpus eritematoso sistémico neuropsiquiátrico em Portugal

RESUMO - Este estudo tem como objetivo principal analisar o impacto do lúpus eritematoso sistémico (LES) na qualidade de vida relacionada com a saúde num grupo de doentes portugueses, através de instrumentos genéricos. Trata-se de um estudo transversal, onde os instrumentos de colheita de dados foram o EQ-5D-3L e o SF-36 v.2, aplicados no decorrer de 2012, sendo a população constituída por 116 doentes com LES-NP, inscritos na Unidade de Doenças Autoimunes do Hospital Curry Cabral, Lisboa. Todos os doentes preencheram os critérios revistos ACR de 97 para a classificação de LES. Dos 116 doentes, 104 eram mulheres (89,7%) e 12 homens (10,3%). A idade média da amostra é de 46 anos. Os indivíduos apresentam um critério ACR médio de 5,0, um índice SLICC/ACR (SDI) médio de 0,6 (± 1,1) e uma duração média de doença de 13 anos, onde 50% dos participantes indica ser portador de LES pelo menos há 11 anos. Os resultados obtidos evidenciaram que a qualidade de vida relacionada com a saúde no LES varia em função de determinadas características demográficas dos doentes como são o caso da idade e o sexo, relacionando-se também com a duração da doença e o índice de irreversibilidade de lesão, nalgumas dimensões do EQ-5D-3L e SF-36. Estes dois instrumentos genéricos apresentam forte associação em todas as suas dimensões. Um achado relevante, prende-se com o facto de o LES apresentar uma pior qualidade de vida relacionada com a saúde em relação a outras doenças crónicas comparáveis.

Frequency of Mycoplasma hominis and Ureaplasma urealyticum infections in women with systemic lupus erythematosus

Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) have been detected in the urine of women with systemic lupus erythematosus (SLE). We evaluated the presence of these mycoplasma in the endocervix of women presenting SLE. A total of 40 SLE patients (mean age 40.2 years), and 51 healthy women (mean age 30.9 years), were studied. Endocervical swabs were cultured in specific liquid media for MH or UU, detected by a quantitative color assay, and considered positive at >10³ dilutions. Statistical analysis was performed using the two-tailed Fisher test. UU was detected in 52.5 % of patients and in 11.8% of controls (p= 0.000059). MH was detected in 20% of patients and 2% controls (p=0.003905). Both mycoplasmas were detected in 7.3% patients and 0% controls (p<0.000001). The results reported here corroborate the association of the mycoplasma infection and SLE. Thus, these agents may stimulate the production of autoreactive clones.

Rastreio combinado do 1º trimestre e doenças autoimunes : Impacto das variaveis pré-analíticas na avaliação do risco

RESUMO: Contexto: As anomalias congénitas, com particular destaque para as neuploidias , afectam aproximadamente 2% dos recém-nascidos, constituindo causas frequentes de morbilidade e mortalidade. Actualmente, a avaliação do grau de risco para as aneuploidias mais prevalentes (T21, T13, T18) é efectuada através do “Rastreio Combinado do 1º Trimestre”, devendo as grávidas com risco acrescido ser sujeitas a exames invasivos (ex.biópsia das vilosidades coriónicas,amniocentese). Quanto mais qualidade existir num rastreio, menos falsos positivos existirão e menor o número de exames diagnósticos invasivos desnecessários. As doenças autoimunes são doenças inflamatórias crónicas em cuja fisiopatologia se encontram distúrbios da imunidade humoral e celular, dependentes de factores genéticos, hormonais,psicológicos e ambientais. Atingem mais o sexo feminino e durante a idade fértil,podendo influenciar o outcome da gravidez e a saúde neonatal causando significativa morbilidade e mortalidade. O lúpus eritematoso sistémico para além de potencialmente afectado pelas alterações imunoendócrinas fisiológicas da gravidez, associa-se frequentemente a problemas de fertilidade. Recentemente, foi sugerido que as anormalidades ocorridas durante a invasão precoce do sinciciotrofoblasto, resultando em deficiente diferenciação, deficiente maturação e diminuição na produção de hCG, poderão ser o mecanismo fisiopatológico primário para as perdas fetais no primeiro trimestre, nos doentes com SLE. A ocorrência de níveis elevados de hCG total e ß-hCG livre no rastreio para despiste de síndrome de Down do segundo e do primeiro trimestre foi assinalada em grávidas portadoras de lúpus, mas a escassez de estudos comprovativos e a pequena dimensão das amostras estudadas constituiu uma limitação significativa na fidedignidade dos resultados obtidos. Objectivos: O estudo teve como objectivos i. estabelecer valores normativos Portugueses e de distribuição para as MoM’s dos parâmetros séricos do primeiro trimestre, por semana de gestação:(PAPP-A e ß-hCG livre), ii. avaliar a influência que as doenças autoimunes têm sobre as MoM’s individuais dos parâmetros bioquímicos PAPP-A e/ou ß-hCG livre, utilizados no rastreio pré-natal combinado do 1º trimestre, e iii. saber se as doenças autoimunes podem condicionar um aumento da taxa de resultados falsos positivos, com consequente aumento do número de amniocenteses. Metodologia: Estudo longitudinal prospectivo, consistindo num rastreio pré-natal combinado de 1º trimestre para pesquisa de aneuploidias, em duas amostras provenientes do Reino Unido (n= 45,854) e de Portugal (n=3122). Foram avaliados parâmetros socio-demográficos, ecográficos, laboratoriais, e calculados os indicadores de desempenho do rastreio combinado. A execução analítica dos testes bioquímicos séricos (PAPP-A e ß-hCG livre) foi realizada no autoanalisador Brahms Kryptor e no autoanalizador 6000 Delfia Xpress. Compararam-se os grupos autoimune e não autoimune das amostras. Resultados: Relativamente às características populacionais, o grupo auto imune tinha valores significativamente superiores nas variáveis idade materna e idade gestacional. Comparando os grupos com e sem doença autoimune, constatou-se a existência de uma elevação das MoM’s da ß-hCG livre nas grávidas com doença autoimune, nomeadamente nos casos de lúpus eritematoso sistémico. Conclusões: os resultado obtidos reforçam a indicação do rastreio combinado do 1º trimestre certificado pela FMF nas grávidas com doenças autoimunes, nomeadamente para as doentes com LES; no entanto, devem ser calculados e introduzidos factores de correcção no algoritmo de risco, de modo a evitar a subida no número de resultados falso-positivos, e consequentemente a sobre- utilização de métodos invasivos.------------ ABSTRACT: Context: Congenital anomalies, with particular reference to aneuploidias, affect approximately 2% of newborns, and are frequent causes of morbidity and mortality. Currently, the risk evaluation for the most prevalent aneuploidias (T21, T13, T18) is carried out through the “combined first trimester screening”, and pregnant women with increased risk are subjected to invasive tests (e.g. villus biopsy done, amniocentesis). The more quality exists in a screening, less false positives exists and fewer unnecessary invasive diagnostic exams. Autoimmune diseases are chronic inflammatory diseases in whose pathophysiology are immune humoral and cellular disorders, dependent on genetic factors, hormonal, psychological and environmental factors. The disease is more prevalent among females, during the child-bearing age, and may influence the outcome of pregnancy and neonatal health causing significant morbidity and mortality. Lupus Erythematosus in addition to potentially affected by immunoendocrine physiological changes of pregnancy, is often associated with fertility problems. Recently, it has been suggested that the abnormalities that occurred during the early invasion of the syncytiotrophoblast, resulting in insufficient differentiation, deficient maturation and decrease in production of hCG may be the primary pathophysiological mechanism for fetal losses in the first quarter, in patients with SLE. The occurrence of elevated levels of total hCG and free ß-hCG in screening for Down’s syndrome of the second and first trimester was reported in pregnant women with lupus, but the paucity of supporting studies and the small size of the samples studied constituted a significant limitation on the trustworthiness of the results obtained. Objectives: this study aims to i. establish normative values for the serum parameters MoM’s (PAPP-A and free β-hCG) and it’s distribution, in the first trimester, by week of pregnancy; ii. assess the influence that the autoimmune diseases have on the MoM’s of individual biochemical PAPP-A and/or β-hCG, used in antenatal screening combined for the first trimester, and iii. whether the autoimmune diseases may make an increased rate of false positives, with consequent increase in the number of amniocenteses.Methodology: Prospective longitudinal study, consisting of a combined first trimester antenatal screening for aneuploidies lookup in two samples from the United Kingdom (n=45.854) and Portugal (n= 3.122). Socio-demographic, echographic and laboratory parameters were evaluated, and combined screening performance indicators were calculated. The analytical run of serum biochemical tests (PAPP-A and ß-hCG) was held at the Brahms Kryptor and in Delfia Xpress 6000. Comparisons between autoimmune group and non-autoimmune group were made. Results: Relating to population characteristics, the autoimmune group had significantly diferente values in the variables maternal age and gestational age. Comparing the groups with and without autoimmune disease, it was noted that there is an elevation of the MoM’s of free ß-hCG levels in pregnant women with autoimmune disease, particularly in cases of systemic lupus erythematosus. Conclusions: The results obtained reinforce the indication of FMF certified combined screening in pregnant women with autoimmune diseases, notably to the patients with SLE; However, correction factors should be calculated and entered in the risk algorithm, in order to avoid the rise in the number of false positive results, and consequently the over-use of invasive methods.

Catatonia due to systemic lupus erythematosus

Objectives Discuss neuropsychiatric aspects and differential diagnosis of catatonic syndrome secondary to systemic lupus erythematosus (SLE) in a pediatric patient. Methods Single case report. Result A 13-year-old male, after two months diagnosed with SLE, started to present psychotic symptoms (behavioral changes, hallucinations and delusions) that evolved into intense catatonia. During hospitalization, neuroimaging, biochemical and serological tests for differential diagnosis with metabolic encephalopathy, neurological tumors and neuroinfections, among other tests, were performed. The possibility of neuroleptic malignant syndrome, steroid-induced psychosis and catatonia was also evaluated. A complete reversal of catatonia was achieved after using benzodiazepines in high doses, associated with immunosuppressive therapy for lupus, which speaks in favor of catatonia secondary to autoimmune encephalitis due to lupus. Conclusion Although catatonia rarely is the initial clinical presentation of SLE, the delay in recognizing the syndrome can be risky, having a negative impact on prognosis. Benzodiazepines have an important role in the catatonia resolution, especially when associated with parallel specific organic base cause treatment. The use of neuroleptics should be avoided for the duration of the catatonic syndrome as it may cause clinical deterioration.

Mitral restenosis in the early postoperative period of a patient with systemic lupus erythematosus

A forty eight year old woman, who had undergone mitral comissurotomy and subsequently developed early restenosis, presented with major comissural fusion and verrucous lesions on the cuspid edges of the mitral valve, with normal subvalvar apparatus. Patient did well for the first six months after surgery when she began to present dyspnea on light exertion. A clinical diagnosis of restenosis was made, which was confirmed by an echocardiogram and cardiac catheterization. She underwent surgery, and a stenotic mitral valve with verrucous lesions suggesting Libman-Sacks' endocarditis was found. Because the diagnosis of systemic lupus erythematosus (SLE) had not been confirmed at that time, a bovine pericardium bioprosthesis (FISICS-INCOR) was implanted. The patient did well in the late follow-up and is now in NYHA Class I .

Echocardiographic Abnormalities and Antiphospholipid Antibodies in Patients with Systemic Lupus Erythematosus

OBJECTIVE: Lupus anticoagulant and anticardiolipin antibodies (aCL) have been associated with thrombosis, recurrent abortion, and thrombocytopenia in patients with systemic lupus erythematosus (SLE), but their relationship with cardiac disease is less clear. The purpose of this study was to evaluate the association between antiphospholipid antibodies (aPL) and echocardiographic abnormalities in patients with SLE. METHODS: A total of 70 consecutive patients and 42 control subjects underwent M-mode, 2-dimensional and Doppler echocardiography and tests for lupus anticoagulant, aCL IgG, IgM, and IgA. Lupus anticoagulant was assayed with the dilute Russell viper venom time, and aCL IgG, IgM, and IgA were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Lupus anticoagulant showed a prevalence of 10%. As a whole, aCL had a prevalence of 44.3% and aPL had a prevalence of 50%. Patients with echocardiographic abnormalities had a prevalence of 54.3% and showed a trend towards an association with aCL IgG (P=0.06). The presence of pulmonary hypertension (PH) was significantly associated with aCL IgG (p=0.02). CONCLUSION: aCL IgG was significantly associated with PH and showed a strong trend towards an association with echocardiographic abnormalities taken together. These findings suggest a role for aCL IgG in the development of lupus cardiovascular disease.

INFLUENCIA DE LAS VARIACIONES ECOLÒGICAS Y AMBIENTALES EN LA EMERGENCIA DE FLAVIVIRUS DE IMPORTANCIA SANITARIA EN LA CIUDAD DE CÓRDOBA

El VSLE, desde el año 2002, reemergió como agente etiológico de encefalitis en humanos en América del Sur, generando casos aislados en Argentina. Córdoba sufrió recientemente la introducción de los virus Dengue y VWN y la emergencia del virus SLE como patógeno causante de encefalitis en humanosel cual sorpresivamente, en el verano-otoño de 2005, produjo por primera vez una epidemia de encefalitis en humanos en Córdoba ciudad, inédita para América del sur. Nuestros estudios permitieron conocer que el ciclo de transmisión estaría integrado por mosquitos Cx quinquefasciatus y Cx. interfor y palomas Zenaida auriculata y Columbina picui.Sobre VWN, estrechamente relacionado a VSLE, los primeros antecedentes de actividad en Argentina son del año 2006. Sin embargo, en poblaciones silvestres de aves, confirmamos por seroprevalencia su actividad desde fines del año 2004. Desconocemos el ciclo del VWN en Argentina, aunque, demostramos preliminarmente que Columbina picui actuaría como hospedador en el ciclo de mantenimiento del virus. Estos resultados similares a los obtenidos para el VSLE indicarían que ambos virus podrían compartir hospedadores aviares en Argentina.Las escasas notificaciones sobre WNV en humanos en el país,muestran registros de casos de encefalitis por este virus en las provincias de Chaco, Córdoba y Entre Ríos en 2006 y en Córdoba, Formosa, y Santa Fe en 2007.En referencia a virus Dengue, en el año 1997 ocurrieron los primeros casos autóctonos de esta enfermedad ( Degue 2), en la provincia de Salta, con un total acumulado de casos hasta el 2007 de 4700. En la epidemia ocurrida recientemente en el 2009 el número de casos confirmados por laboratorio y/o nexo epidemiológico superó los 25.000.El virus DEN, junto con los virus SLE y WN co-circulan en tiempo y espacio en las regiones centrales y norte de nuestro país, generando complicaciones a la hora del diagnóstico de casos febriles.Los factores que provocaron la re-emergencia del VSLE y emergencia de los virus DEN y WN en la región central de Argentina se desconocen al presente. La variabilidad climática ligada a ENSO ( El NIño / Southern oscillation) ha demostrado incrementar las enfermedades de transmisión vectorial tales como encefalitis por Murray Valley, malaria, bluuetongue .Considerando que el cambio en las condiciones climáticas y el uso de la tierra observadas en la ciudad de Córdoba y zona rural del arco sur de la laguna de Mar Chiquita podrían influir en la actividad espacio-temporal de los virus DEN, SLE y WN, el objetivo de proyecto es determinar la influencia de las variaciones climáticas y modificaciones ambientales sobre la actividad de flavivirus de importancia sanitaria en las zonas seleccionadas de la provincia de Córdoba. Se generará una Base de Datos obtenidos sobre mosquitos ,aves y metereológicos retrospectivos y prospectivos que serán sometidos A Diversos Análisis Estadísticos. Se desarrollará y aplicará un SIG Para El Estudio De La Influencia De Modificaciones Ambientales Y Climáticas Sobre La Emergencia De Flavivirus. Se Realizarán Inoculaciones Experimentales En Aves Domésticas Y Silvestres A Fin De Evaluar Su Rol Como Hospedadores. Se Realizarán Estudios Experimentales De competencia Vectorial De Aedes Aegypti, Culex Interfor Y Cx. Quinquefasciatus.Se estudiaran factores ambientales y ecológicos de criaderos de Cx quinquefasciatus En La Ciudad De Córdoba Que Afectan Sus Dinámicas Poblacionales Y Factibles De Ser Aplicados En El Diseño De Planes De Vigilancia De Los Virus Sle Y Wn Y Control De Vectores.Así También Se Desarrollarará Un Modelo Para Predecir Posibles CriadeMediante El Uso De Sensores Remotos Satelitales Se Estudiaran Las Fluctuaciónes Espacio-Temporal De Ae. Aegypti.El proyecto brindará la información necesaria para analizar el rol de las variaciones por el cambio climático que sufre la región en la emergencia de enfermedades virales endémicas de transmisión vectorial.

Mosquitos no Parque Nacional da Serra dos Orgãos, Estado do Rio de Janeiro, Brasil: II. Distribuição vertical

Em prosseguimento aos estudos sobre a ecologia de culicídeos que vimos realizando no Parque Nacional da Serra dos Orgãos (PNSO), apresentamos nesta oportunidade a sua distribuição vertical. Por meio de capturas feitas em isca humana concomitantemente ao nível do solo e nas imediações da copa das árvores, estabelecemos as tendências das espécies que ali ocorreram de março de 1981 a fevereiro de 1982, por se alimentarem de sangue na copa da floresta ou junto ao solo. A distribuição é analisada comparativamente em ambos os níveis levando-se em consideração as variações de temperatura, umidade, precipitações pluviométricas e estações do ano. Dentre as espécies com nítida preferência à acrodendrofilia encontramos alguns importantes transmissores de doenças: Anopheles cruzii - malária humana e simiana; Culex nigripalpus - encefalite de São Luis (SLE); Haemagogus leucocelaenus e Haemagogus capricornii - febre amarela silvestre, todas também sendo obtidas, embora em menor número, a nível do solo. Os sabetíneos - Wyeomyia Knabi, Phoniomyia theobaldi, Sabethes tarsopus, Sabethes quasicyaneus, Sabethes chloropterus - completam a relação das principais espécies que preferem a copa. Por outro lado, Aedes fluviatilis, Trichoprosopon digitatum, Tr. similis, Tr. frontosus, Tr. theobaldi, Wy, arthrostigma, Wy. aporonoma, Wy. personata, Wy undulata, Wy. mystes, Limatus durhami, Li. pseudomethisticus, Sa. identicus e Sa. undosus foram capturados em grande maioria próximo ao solo.