Este trabalho apresenta o processo para obtenção de partículas de sílica amino-funcionalizadas contendo complexos de TR3+ utilizando os métodos de Stöber e por micro-ondas. Os espectros de absorção no infravermelho das partículas TR-BTC-Si preparadas pelo método de micro-ondas exibiram bandas de absorção atribuídas aos modos vibracionais dos complexos TR-BTC e da rede de sílica, indicando a incorporação destes complexos na matriz SiO2. Por outro lado, os complexos Eu-(β-dicetonatos) preparados pelo método Stöber mostraram apenas as bandas atribuídas à estrutura da rede de sílica, devido à dupla camada de revestimento de sílica. As morfologias das partículas de sílica amino-funcionalizadas contendo complexos de TR3+ foram visualizadas usando a técnica MEV. As diferenças nas morfologias entre o complexo precursor e o material amino-funcionalizado pode ser atribuído a presença da sílica na superfície do material. Além do mais, o método da ninidrina indicou a presença de grupos amina (-NH2) na superfície destes materiais. Os espectros de emissão dos materiais funcionalizados com os complexos de Eu3+ e Tb3+ apresentaram as bandas de emissão da transição intraconfiguracional dos íons Eu3+ (5D0→7FJ, J = 0-6) e Tb3+ (5D4→7FJ, J = 6-0), exibindo cores características de emissão vermelha e verde, respectivamente. É observado um decréscimo nos valores dos parâmetros Ω2 dos materiais Eu-(β-dicetonato)-Si e Eu-BTC-Si comparados com os respectivos complexos, devido a diminuição da intensidade da transição 5D0→7F2, indicando um maior caráter centrossimétrico. Conseqüentemente, os íons Eu3+ nos materiais com sílica estão em um ambiente químico menos polarizável do que nos complexos, sugerindo uma menor contribuição do mecanismo de acoplamento dinâmico. Os altos valores dos Ω4 para os sistemas com sílica Eu-(&#946-dicetonato)-Si e Eu-BTC-Si comparados com os valores de Ω2 reflete a intensidade extremamente alta da transição 5D0→7F4 observada nos espectros de emissão. Este resultado corrobora com o maior caráter centrossimétrico e evidencia a incorporação dos complexos de Eu3+ na rede de sílica. Foi realizado um fluoroimunoensaio usando os compostos amino-funcionalizados, que mostraram uma luminescência e propriedades físico-químicas eficientes para atuarem como marcadores biológicos. O marcador óptico foi conjugado com o anticorpo anti-oxLDL, que se liga a um suporte específico com o antígeno oxLDL. Portanto, estes materiais são candidatos promissores para conjugação molecular em clínicas de diagnóstico. / This work presents the process to obtain amino-functionalized silica particles containing complexes of trivalent rare earth ions (RE3+) using Stöber and microwave methods. Infrared spectra of the TR-BTC-Si particles prepared by microwave method exhibited absorption bands assigned to the vibrational modes of the TR-BTC complexes and silica network, indicating that the complexes have been incorporated in the SiO2 matrix. However, due to double coating of the silica network, the IR spectra of the Eu-(β-diketonates) complexes prepared by Stöber method showed only bands assigned to the silica structure. The morphologies of the amino-functionalized silica particles containing RE3+ complexes were examined using SEM technique. The difference in morphology between the complex precursor and amino-functionalized material can be attributed to the silica network on the material surface. Besides, the ninhydrin method confirmed the presence of amine groups (-NH2) in the functionalized materials. The emission spectra of the functionalized materials containing Eu3+ and Tb3+ complexes showed the emission bands originated from the intraconfigurational transitions of the Eu3+ (5D0→7FJ, J = 0-4) and Tb3+ (5D4→7FJ, J = 6-0), exhibiting red and green color emission, respectively. It is observed decreasing values of experimental intensity parameters (Ω2) of the Eu-(β-diketonate)-Si and Eu-BTC-Si materials when compared with the complex precursors, owing to the decreased intensity of the 5D0→7F2 transition, indicating a higher centrosymmetric character. As a result, the Eu3+ ions in silica materials are located in a chemical environment less polarizable than in the complexes, suggesting a smaller contribution of dynamic coupling mechanism. On the other hand, an abnormally high intensity of the 5D0→7F4 transition was observed, which is reflected by the high values of Ω4 of the silica systems, Eu-(β-diketonate)-Si and Eu-BTC-Si, compared with the Ω2 ones. These spectroscopic data corroborate with a higher centrosymmetric character, indicating the incorporation of the Eu3+ complexes in the silica network. A fluoroimmunoassay was developed using the amino-functionalized compounds that exhibit efficient luminescence and physical and chemical properties suitable for optical label. The biolabel was then chemically conjugated to anti-oxLDL antibody, which is linked in a specific support with oxLDL antigen. The result showed that it is a promising candidate for molecular conjugation in clinical diagnosis.

Introdução: a vasculopatia livedoide é uma doença cutânea rara que acomete principalmente mulheres adultas, cursando inicialmente com lesões purpúricas e necróticas intensamente dolorosas, acometendo extremidades inferiores, que podem evoluir com úlceras de tamanhos variados. Estas lesões costumam evoluir com cicatrizes atróficas, estreladas e com telangiectasias, denominadas cicatrizes de atrofia branca. Apesar da etiologia não estar totalmente esclarecida, os distúrbios da coagulação parecem ser o mecanismo fisiopatológico primário da vasculopatia livedoide. Inúmeras trombofilias já foram associadas à vasculopatia livedoide, porém cerca de metade dos casos permanece sem etiologia definida. Recentemente foi documentado aumento de lipoproteína(a) na vasculopatia livedoide. Níveis plasmáticos elevados de lipoproteína(a) são considerados um fator de risco causal independente para o desenvolvimento de doenças cardiovasculares e trombóticas, devido à sua similaridade estrutural com o plasminogênio. A deposição tecidual da lipoproteína(a) em vasos ateroscleróticos é bem descrita, porém na vasculopatia livedoide a participação da lipoproteína(a) não é conhecida. Objetivos: analisar os níveis séricos da lipoproteína(a) e a sua expressão tecidual na pele dos pacientes com vasculopatia livedoide e comparar os resultados com um grupo controle. Métodos: amostras de pele obtidas através de biópsia de pele de 38 pacientes com vasculopatia livedoide (27 do sexo feminino e 11 do sexo masculino) e 9 indivíduos do grupo controle (5 do sexo feminino e 4 do sexo masculino) foram avaliados para a presença de lipoproteína(a) através de imuno-histoquímica utilizando anticorpo policlonal anti- lipoprotéina(a). Os níveis plasmáticos da lipoprotéeíina(a) foram analisados por imunoturbidimetria e comparados com setenta pacientes portadores de outras doenças dermatológicas. Resultados: utilizando anticorpo policlonal contra lipoproteína(a), observou-se que a pele perilesional em pacientes com vasculopatia livedoide apresentava dez vezes mais lipoprotéina (a) do que a pele controle. O coeficiente de correlação de Pearson (r = 0,02) mostrou que os níveis teciduais de lipoproteína(a) não se correlacionaram com os níveis plasmáticos nos pacientes com vasculopatia livedoide. Níveis plasmáticos elevados de lipoproteína(a) foram observados nos portadores de vasculopatia livedoide, no entanto não pode ser demonstrada diferença estatística em relação ao grupo controle. Conclusões: achados de aumento da expressão tecidual de lipoproteína(a) na pele lesionada e níveis plasmáticos elevados desta proteína em pacientes com vasculopatia livedoide podem corroborar com a hipótese de que a lipoproteína(a) possa contribuir para a patogênese da vasculopatia livedoide através dos seus efeitos antifibrinolíticos, pró- trombóticos e ação direta no endotélio vascular, induzindo a formação do trombo / Background: livedoid vasculopathy is a chronic disorder that usually presents as recurrent reticulated purpura on the lower limbs, with recurrent painful, purpuric and/or necrotic macules that may lead to ulcerative lesions. These lesions usually heal into atrophic white scars, with depigmentation and telangiectasias, known as \"atrophie blanche\". Although the etiology of the livedoid vasculopathy is not fully understood, coagulation disorders appear to be the primary pathophysiological mechanism. Hereditary thrombophilia has been associated with livedoid vasculopathy, but about half of the cases remain without defined etiology. Recently, high serum levels of lipoprotein(a) in livedoid vasculopathy patients have been documented. Elevated plasma levels of lipoprotein(a) are an independent risk factor for the development of cardiovascular and thrombotic diseases due to their structural similarity with plasminogen. Lipoprotein(a) deposition in atherosclerotic vessels is well described, but its role in livedoid vasculopathy is unknown. Objectives: To analyze the serum levels of lipoprotein(a) and tissue expression in the skin of patients with livedoid vasculopathy and to compare the results with a control group. Methods: Skin biopsy samples from 38 patients (27 women-11 men) with active lesions diagnosed with livedoid vasculopathy and 9 samples of normal skin (5 women-4 men) from control individuals without livedoid vasculopathy were evaluated for skin expression of lipoprotein(a) by immunohistochemistry using polyclonal anti-lipoprotein(a) antibody. Plasma levels of lipoprotein(a) were analyzed by immunoturbidimetry and compared with seventy patients with other dermatological diseases. Results: We found lesional skin in patients with livedoid vasculopathy expressed tenfold higher lipoprotein(a) immunostaining than controls. High plasma levels of lipoprotein(a) were observed in livedoid vasculopathy patients, but we cannot observed a positive correlation (p = 0.02) between skin expression of Lipoprotein(a) and plasma levels of Lipoprotein(a) in patients with livedoid vasculopathy. Conclusions: Increased of lipoprotein(a) tissue expression on lesioned skin and elevated plasma levels of this protein in patients with livedoid vasculopathy may corroborate the hypothesis that lipoprotein(a) may contribute to the pathogenesis of livedoid vasculopathy through its antifibrinolytic effects, prothrombotic and direct action on the vascular endothelium, inducing thrombosis

Alta concentração de lipoproteína modificada de baixa densidade (LDL) é um dos fatores que aumentam o risco de doenças cardíacas. As concentrações mais elevadas de LDL modificada estão associadas à aterosclerose nas veias de sangue. A técnica de Z- scan (ZS) foi utilizada para investigar a resposta óptica não-linear da lipoproteína modificada de baixa densidade (moLDL). As características estruturais do mLDL foram estudadas por espalhamento dinâmico de luz. Nesta pesquisa, os experimentos foram realizados em amostras de LDL de 24 pacientes com diabetes e gengivite (grupo 1) e 24 pacientes com diabetes e periodontite (grupo 2). Os experimentos foram repetidos após 6 meses sem qualquer tratamento para o grupo 2 e com um tratamento periodontal para o grupo 1. Os resultados mostram que a amplitude da curva Z-scan no grupo 1 é menor que a do grupo 2. Após 6 meses de tratamento periodontal a amplitude é maior do que antes, depois a concentração de LDL modificado é reduzida nos pacientes tratados. Portanto, o risco cardiovascular para aterosclerose é diminuído após o tratamento periodontal. Além disso, os resultados do espalhamento dinâmico de luz mostram que não há nenhuma mudança estrutural significativa no moLDL entre os dois grupos antes e após 6 meses. / High-concentration of modified low-density lipoprotein (LDL) is one of the factors that increases the risk of heart disease. The higher concentrations of modified LDL are associated with the atherosclerosis in the bloodstream. The Z-scan (ZS) technique was used to investigate the nonlinear optical response of modified low-density lipoprotein (moLDL). The structural characteristics of moLDL were studied by dynamic light scattering. In this work, the experiments were carried out in LDL solution from 24 patients with diabetes and gingivitis (group 1) and 24 patients with diabetes and periodontitis (group 2). The experiments had been repeated after 6 months without any treatment for group 1 and with a periodontal treatment for group 2. The results show that the amplitude of Z-scan curve in group 2 is lower than that of group 1. After 6 months of periodontal treatment the amplitude is getting higher than before, then the concentration of modified LDL is reduced in the patients treated. Therefore, the cardiovascular risk for atherosclerosis is decreased after the periodontal treatment. Also, dynamic light scattering results show that there is not a significant structural change in moLDL between the two groups before and after 6 months.

Em trabalhos anteriores, mostrou-se que uma nanoemulsão lipídica denomi-nada LDE após injeção endovenosa, em pacientes com carcinoma mamário e outros tumores sólidos concentra-se nos tecidos neoplásicos e pode direcionar especificamente agentes quimioterápicos ao tumor. Estudos clínicos mostraram que a LDE diminui acentuadamente os efeitos tóxicos desses agentes e em estudos com animais de experimentação não reduz seus efeitos antitumorais. No presente estudo, testamos a hipótese de a LDE injetada por via locorregional poderia concentrar-se no tumor de mama e nos linfonodos axilares da mama comprometida, visando futuras aplica-ções do sistema na quimioterapia neoadjuvante desse tumor. Três técnicas de injeção da LDE foram testadas em pacientes com carcinoma de mama avançado com tratamento cirúrgico pré-programado. A LDE marcada com colesterol radioativo foi injetada 12 horas antes da cirurgia, nas pacientes divididas em três grupos: Grupo 1 (G1, n=4): LDE injetada no parênquima mamário, a 5 cm da lesão ; Grupo 2 (G2, n=4): LDE injetada na região peri-tumoral; e Grupo 3 (G3 n=6): LDE injetada em região intratumoral. Este grupo foi subdividido em 2: em 2 pacientes realizaram cirurgia 2hs após a injeção da LDE e nas outras 4, 12hs após a injeção da LDE. Quantificou-se a capta-ção da LDE nos fragmentos de tecido tumoral e mamário normal e do linfonodo axilar retirados durante a cirurgia por contagem de radioatividade após extração lipídica dos tecidos. Os resultados evidenciaram que, em G1, houve maior captação da LDE em tecido normal, sugerindo se tratar de metodologia ina-dequada. Em G2, a captação da LDE foi quatro vezes maior no tecido tumo-ral do que no tecido normal, próximo, portanto, dos valores encontrados em estudos anteriores que utilizaram a injeção da LDE por via hematológica. Em G3, o valor médio de captação da LDE foi 53 vezes maior no tecido tumoral (>75%), com mínima captação pelo tecido mamário normal (<8%). A injeção intratumoral da LDE mostrou-se, portanto, metodologia ainda superior à inje-ção da LDE por via endovenosa para a utilização efetiva dessa nanoemulsão como terapia-alvo em tumores primários de mama. / In previous work had showed that a lipidic nanoemulsion called LDE after intravenous injection in patients with breast carcinoma and others solid tumors focuses in the neoplasic tissue and can target especially chemotherapist agents to the tumors. Clinical studies showed that LDE decreases the toxic effects of those agents and in studies with experimental animals don\'t reduce yours antitumoral effects. In the present study, we had test the hypothesis of the LDE injected by locoregional could focuses in the breast tumor and in the axillary lymph node of the compromised breast, targeting future applications of the system in the neoadjuvant chemotherapy of this tumor. Three techniques of injection of the LDE had test in patients with advanced breast carcinoma with sirurgic treatment pre-programmed. The LDE marked with radioactive cholesterol was injected 12 hours before the surgery, in patients divided in three groups: Group 1 (G1, n=4): LDE injected in the breast parenchyma, about 5 cm of the lesion; Group 2 (G2, n=4): LDE injected in the peritumoral region; and Group 3 (G3, n=6): LDE injected in the intratumoral region. This group was subdivided in 2: in 2 patients had realized surgery 2 hs after the injection of LDE and in the others 4 patients 12hs after the injection of LDE. It had quantified the capture of LDE in the fragments of the tumoral and breast tissue and of the axillary lymph node withdrawn during the surgery by counting of radioactive after lipidic extraction of this tissue. The results showed that, in G1, there was greater capture of LDE in normal tissue, suggesting it is an inappropriate methodology. In G2, the capture of LDE was four times higher in the tumoral tissue than in the normal tissue, closer, therefore, of the values found in previous studies that used the injection of LDE by hematology. In G3, the medium value of capture of LDE was 53 times higher in the tumoral tissue (> 75%), with minimum capture by the normal breast tissue (< 8%). The intratumoral injection of LDE showed, therefore, methodology still higher than the intravenous injection of LDE by intravenous for the effective use of this nanoemulsion as therapy target in primary tumors of breast.

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in nitric oxide (NO) production by macrophages. A major goal of this study was to identify the lipid components in oxLDL modulating NO production. The effect of a water soluble antioxidants (N-acetylcysteine) and lipid soluble antioxidant (alpha-tocopherol) on NO production in macrophages was also determined. A second goal was to determine if the effects of oxLDL occurred at the transcriptional level. Human LDL was oxidized using an azo-initiator 2,2$\sp\prime$-azobis (2-amidinopropane) HCI (ABAP). OxLDL markedly decreased the production of NO in LPS stimulated RAW264.7 macrophages. This inhibition depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. In contrast, acetylated LDL (AcLDL) and native LDL showed only minor inhibition. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the primary products formed during LDL oxidation. 13-Hydroperoxyl octadecadienoic acid (13-HPODE) markedly inhibited NO production, whereas lysoPC showed only slight inhibition. Furthermore, the effects of 13-HPODE and lysoPC did not require their uptake in an AcLDL carrier. Pre-treatment of macrophages with alpha-tocopherol attenuated the inhibition due to oxLDL. Similarly, pre-treatment with N-acetylcysteine attenuated the inhibition caused by oxLDL or 13-HPODE. OxLDL was found to decrease iNOS protein and mRNA levels in RAW264.7 macrophages induced by LPS. The activation of NF-$\kappa$B was slightly suppressed after 45 minutes of treatment. 13-HPODE showed much stronger reduction of iNOS protein levels than lysoPC. These results suggest that oxLDL may inhibit NO production in macrophages at transcriptional level. 13-HPODE is likely to be the most important lipid component in oxLDL for the inhibitory effect. Antioxidants were found to preserve NO production in macrophages treated with either oxLDL or 13-HPODE. The physiological consequences of decreased NO production in macrophages caused by oxLDL are discussed with respect to atherosclerosis.

Lysophosphatidic acid (LPA) is a potent bioactive lipid component of oxidized low density lipoproteins (oxLDL). High concentrations of LPA have been detected in human atherosclerotic plaques. Our data has shown that LPA highly induces smooth muscle cell (SMC) migration. Cyr61, a matricellular protein, which also accumulates in human atherosclerotic plaques, has been implicated in the injury-induced neointimal formation. Smooth muscle cell migration is a key event in the development of atherosclerosis, and it contributes to the progressive growth of atherosclerotic lesions. Data generated by this study demonstrate that LPA markedly induces Cyr61 expression in mouse aortic smooth muscle cells (MASMC). We hypothesized that LPA-induced matricellular Cyr61 mediates LPA-induced MASMC migration. To date, little is known about the relationship between LPA and Cyr61 in smooth muscle cells; the signaling pathway leading to LPA-induced Cyr61 is unknown. Furthermore, whether Cyr61 contributes to LPA-induced cell migration is unrevealed. Our study demonstrates that LPA, by binding to LPA1 receptor, activates the intracellular signaling pathway leading to the activation of PKCdelta which in turn contributes to the increased expression of Cyr61 in MASMCs. Interestingly, we found that after LPA-induced Cyr61 mRNA has been translated into its protein intracellularly, the de novo synthesized proteins promptly accumulate in the Golgi apparatus and then translocalize to the extracellular matrix. Importantly, our data reveal a novel LPA/Cyr61 pathway in controlling MASMC migration. Understanding the mechanism underlying LPA induction of Cyr61 provides new insight into pathogenesis of atherosclerosis.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden. We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation. When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate. To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array. A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism. The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

The development of a first myocardial infarction is associated with a large number of contributing factors. Age, male sex, hypertension, smoking, diabetes, body mass index and hypercholesterolemia are considered as established risk factors. The primary aim of the present dissertation was to evaluate whether specific biomarkers could improve the prediction of subjects at risk for a first myocardial infarction when considered in addition to established cardiovascular risk factors. The biomarkers investigated include: tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), thrombomodulin (TM), von Willebrand factor (VWF), dehydroepiandrosterone sulfate (DHEAS), lipoprotein (a) (Lp(a)), leptin, apolipoproptein A1 (ApoA1), proinsulin, homocysteine and homozygosity for the 5,10- methylenetetrahydrofolate reductase (MTHFR) C&gt;T genotype. A secondary objective was to determine whether a first myocardial infarction leads to increased plasma homocysteine concentrations and whether the association between homocysteine and myocardial infarction was greater at follow-up compared to baseline. The study population consisted of 36 405 subjects screened and included in the Västerbotten Intervention Program and the Northern Sweden MONICA cohorts between January 1, 1985 and September 30, 1994. A nested incident case-referent study design was used. Seventy eight cases with a first myocardial infarction were identified, and from the same cohort twice as many sex and age matched referents were randomly selected. Moreover, a follow-up health survey (average 8.5 years between surveys) was conducted with 50 cases and 56 matched referents. High plasma levels of tPA and PAI-1 mass concentration, VWF, proinsulin, leptin and Lp(a) and low plasma levels of ApoA1 were associated with subsequent development of a first myocardial infarction in univariate conditional logistic regression analysis. For PAI-1 and tPA, this relation was found in both men and women. For tPA, but not for PAI-1 and VWF, this association was independent of established risk factors. In women, high plasma concentrations of TM were associated with significant increases in risk of a first myocardial infarction. No predictive values of DHEAS, homocysteine or for the point mutation C677&gt;T in the gene for MTHFR was found regarding the risk of a first myocardial infarction. The summarised importance of haemostatic and metabolic variables (proinsulin, lipids including Lp(a) and leptin) in predicting first myocardial infarction in men, as well as possible interactions among these variables, were studied. High tPA and Lp(a) and low ApoA1 remained significant risk markers in multivariate analysis independent of established risk factors. There were non-significant synergic interactions between high Lp(a) and leptin and tPA respectively, and between high Lp(a) and low ApoA1. In the follow-up study plasma homocysteine and plasma creatinine increased significantly, and plasma albumin decreased significantly over time. Conditional univariate logistic regression indicated that high homocysteine at follow-up but not at baseline was associated with first myocardial infarction but the relation disappeared in multivariate analyses including plasma creatinine and plasma albumin. High plasma creatinine remained associated with first myocardial infarction at both baseline and follow-up. In conclusion, the present results support the hypothesis that biomarkers, in addition to the traditional cardiovascular risk factors, carry predictive information on the risk of developing a first myocardial infarction.

Background: Numerous studies have shown that estrogen replacement therapy (ERT) is an effective treatment for vasomotor symptoms, insomnia and vaginal dryness. Beneficial effects have also been shown on lipid patterns and on the incidence of osteoporotic fractures. As ERT increases the risk of endometrial adenocarcinoma, combinations with various progestogens have been developed in order to protect the endometrium. However, the addition of progestogens tends to reduce the beneficial effects of estrogens on mood, cognition and lipid metabolism. The added progestogen often causes side effects such as irritability and depression. There is evidence that the effect on wellbeing varies between women and with the type of progestogen used. Women who prefer to avoid withdrawal bleedings can be given continuous combined hormone replacement therapy (HRT). Unfortunately, irregular bleedings are common at the beginning of treatment and reduces compliance. Recently, several studies have reported an increased risk of breast cancer and venous thrombosis, and therefore long-term treatment with HRT for women without climacteric symptoms is no longer recommended. The ongoing debate has, for the time being, resulted in a recommendation that improving quality of life (QoL) by treatment of climacteric symptoms should be the only indication for prescribing HRT. Aims and methods: The aims of the study were to investigate bleeding patterns, changes in wellbeing at onset and during long-term treatment, and lipid and lipoprotein profiles with two different types of continuous combined HRT. In addition, women starting, and women switching from mainly sequential HRT were compared. The design was a randomised, double-blind, one year, prospective, multicentre study including 249 healthy postmenopausal women who were given continuous daily oral treatment with either combined 0.625mg conjugated estrogen (CE) and 5mg medroxyprogesterone acetate (MPA) or combined 2mg 17β - estradiol (E2) and 1mg norethisterone acetate (NETA). Bleedings, if any, were recorded daily throughout the study. The main outcome measures (changes in wellbeing and climacteric symptoms) consisted of daily ratings of 12 items on a validated symptom scale. Serum concentrations of lipids and lipoproteins were measured at baseline and after one year of treatment. Results and conclusions: The majority of drop-outs were confined to the first three months, and the main reasons were bleedings and/or decreased wellbeing. Drop-outs were three times more common in the E2/NETA group. During the first month, 67% of the women reported irregular bleedings. The number of bleeding days decreased on both treatments during the first four months. Treatment with CE/MPA resulted in less irregular bleedings and a shorter time to amenorrhoea compared to E2/NETA. As expected, "starters" experienced more sweats than "switchers" at the onset of treatment, but both groups improved significantly. Side effects such as breast tenderness, swelling, depression and irritability appeared during the first treatment week in both groups. The side effects of HRT appeared much more quickly than the benefits and were more frequent in women with a history of premenstrual syndrome (PMS). Breast tenderness was more common in the E2/NETA group throughout the whole study period. Apart from that, there were no differences between the two treatment regimens as regards effects on well-being at the end of the study. Lipoprotein(a) levels, an important risk factor for cardiovascular disease, decreased in both treatment groups. Triglyceride levels increased in women treated with CE/MPA, and levels of total cholesterol, high density lipoprotein and low density lipoprotein fell in the E2/NETA group. In conclusion, treatment with E2/NETA caused more bleeding problems than treatment with CE/MPA. CE/MPA was better tolerated than E2/NETA at the beginning of the study, but among the women remaining in the study there was no difference in QoL between the two treatment groups. HRT counselling should take into account that a history of PMS increases the likelihood of side effects and that these may precede any beneficial effects. Both treatments produced beneficial effects on lipid and lipoprotein levels, and neither of the regimens was superior in this respect.

Cryopreservation of germplasm is widely used in agriculture, biotechnology, conservation of threatened species and human reproductive medicine. There is a need however to improve the reproductive efficiency of breeding with cryopreserved semen, which may involve increasing the post-thaw quality of sperm through improvements in cryopreservation extenders. Extenders including egg yolk from chickens are successfully used worldwide for cryopreservation of bovine semen, whereas the protective agent in the egg yolk is believed to be the low-density lipoprotein (LDL) fraction. Egg yolks of different avian species vary in their cholesterol, phospholipid and polyunsaturated fatty acid content which have been shown to have important effects on sperm’s freezing capability. The purpose of this study was to determine the cryoprotective effect of clarified egg yolk and LDLs extracted from different egg yolk sources (chicken, chicken omega-3, pigeon, quail and turkey) on bovine sperm. Semen from six bulls was collected four times each by electroejaculation, split and diluted with the 10 following extenders: chicken clarified (Ccl), chicken omega-3 clarified (O3cl), pigeon clarified (Pcl), quail clarified (Qcl), turkey clarified (Tcl), chicken LDL (CLDL), chicken omega-3 LDL (O3LDL), pigeon LDL (PLDL), quail LDL (QLDL) and turkey LDL (TLDL). The extended semen was evaluated, cryopreserved and examined directly after thawing (0h) and after two hours at 37 ˚C (2h). Computer assisted sperm analysis (CASA) was used to determine total sperm motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). Intact plasma membrane (IPM) and intact acrosomes (IA) were measured by flow cytometry. The percentage change (loss; Δ%) of each sperm characteristic was calculated and used to compare the effect of the extenders. From extending to 0h post-thaw, the pigeon LDL extender lead to greater losses in sperm total and progressive motility, as well as of intact acrosomes, than the other nine extenders tested (P < 0.05). During 0h to 2h post-thaw, the sperm in PLDL extender experienced greater losses in total and progressive motility (P < 0.0001), as well as in curvilinear velocity (P < 0.05), than in all the other nine extenders. Sperm in turkey clarified extender had a greater loss in the velocity parameters (VSL, VAP, VCL) than sperm in several of the other extenders such as O3cl, CLDL, O3LDL, QLDL and TLDL from 0h to 2h (P < 0.05). Concomitantly, sperm in the Tcl extender had a greater loss in the velocity parameters and of intact acrosomes compared to sperm in its counterpart, the turkey LDL extender, from 0h to 2h post-thaw (P < 0.05). The differences produced in post-thaw quality of cryopreserved bovine sperm in the pigeon LDL and turkey clarified extenders were attributed to methodological differences in these egg yolk preparations compared with the other eight extenders. Importantly, the results demonstrate that with most egg yolk preparations derived from a variety of species, there are equivalent cryoprotective effects afforded by the use of omega-3 chicken, pigeon, quail, or conventional chicken egg yolk in a clarified form in freezing extenders for bovine semen. We further proved that the freezing capabilities of bovine semen extenders containing the low-density lipoprotein fraction of omega-3 chicken, quail, turkey and conventional chicken egg yolk were similar.