Introdução: A Hipercolesterolemia Familiar (HF) é uma doença genética caracterizada clinicamente por elevados níveis de lipoproteína de baixa densidade (LDL-C) na corrente sanguínea desde a infância. Indivíduos que apresentam HF podem desenvolver doença aterosclerótica ainda em idade jovem. Os principais preditores de risco no desenvolvimento da doença cardiovascular (DCV) nesses indivíduos após entrarem em um programa de rastreamento genético não são conhecidos na nossa população. Além disso, a HF é subdiagnosticada e subtratada mundialmente e o rastreamento genético em cascata dos familiares tem sido mundialmente avaliado como o método diagnóstico mais custo. Contudo, a efetividade do rastreamento genético em cascata é dependente dos critérios clínicos de entrada do primeiro indivíduo da família e não há um consenso de qual critério apresenta a melhor acurácia para detecção de uma mutação. Objetivos: Identificar os fatores determinantes para ocorrência de eventos cardiovasculares (CV) em todos os indivíduos da coorte e avaliar o critério clínico para detecção de uma variante genética patogênica para HF, no primeiro indivíduo da família, após serem inseridos em um programa de rastreamento genético em cascata.Métodos: Estudo de coorte prospectiva aberta dos pacientes que foram inseridos no programa de rastreamento genético em cascata para HF. A população do estudo é definida como caso índice (CI), o primeiro da família a ser identificado clinicamente e encaminhado para o teste genético, e os familiares, que são os parentes de 1º grau do CI em que foi encontrada uma alteração genética. Todos os indivíduos são inseridos na coorte no momento em que recebem o laudo genético (tempo zero, T0). Um ano depois do T0 é realizado o primeiro contato telefônico, ou seja, primeiro ano de seguimento (T1) Resultados: No T1, o total de 818 indivíduos foi incluído, sendo verificados 47 eventos CV, sendo 14 (29,7%) fatais. Para o CI, o único fator independente associado ao aumento do risco de eventos CV no T1 foi a presença de arco corneano (OR: 9,39; IC 95%: 2,46-35,82). Para os familiares com uma mutação positiva os fatores associados ao aumento do risco de eventos CV foram diabetes mellitus (OR: 7,97; IC 95%: 2,07-30,66) e consumo de tabaco (OR: 3,70; IC 95%: 1,09-12,50). Na análise do melhor critério clínico para detecção de uma mutação patogênica no CI os valores de LDL-C >= 230 mg/dL tiveram a melhor relação entre sensibilidade e especificidade. Na análise da curva ROC o escore Dutch Lipid Clinic Network (DLCN) apresentou melhor desempenho do que o LDL-C para identificar uma mutação, a área sob a curva ROC foi 0,744 (IC 95%: 0,704-0,784) e 0,730 (IC 95%: 0,687-0,774), respectivamente, p = 0, 014. Conclusão: Em um ano de seguimento essa coorte identificou uma alta incidência de eventos CV após a entrada em um programa de rastreamento genético em cascata e os preditores dos eventos CV diferem entre CI e familiares. Esses resultados podem contribuir para o desenvolvimento de ações preventivas nesse grupo altamente susceptível de indivíduos. Além disso, devido a importância da detecção da mutação para um diagnóstico definitivo de HF e a importância da cascata ser custo efetiva o estudo identificou que o critério único do LDL-C >= 230 mg/dl é viável para indicar o CI para o teste genético / Introduction: Familial Hypercholesterolemia (FH) is a genetic disease characterized clinically by high levels of low density lipoprotein (LDL-C) in the bloodstream since childhood. Individuals with FH can develop atherosclerotic disease at a young age. The main predictors of cardiovascular disease (CVD) risk in these individuals after entering a genetic screening program are not known in our population. In addition, FH is underdiagnosed and undertreated worldwide and cascaded genetic screening of family members has been evaluated globally as the most cost effective for the diagnosis of FH. However, the effectiveness of cascading genetic screening is dependent on the clinical entry criteria of the first individual in the family and there is no consensus as to which criterion shows the best accuracy for detecting a mutation. Objectives: To identify the determinant factors for cardiovascular (CV) events in all individuals in the cohort and to evaluate the clinical criteria for detecting a genetic variant pathogenic to FH in the first individual of the family after being inserted into a genetic screening program in cascade. Methods: Open prospective cohort study of patients who were enrolled in the cascade genetic screening program for FH. The study population is defined as index case (IC), the first of the family to be clinically identified and referred to the genetic test, and relatives, who are the first-degree relatives of the IC in which a genetic alteration was found. All individuals are inserted into the cohort at the moment they receive the genetic report (time zero, T0). The first follow-up telephone contact is made one year after T0 (first year of follow-up, T1). Results: In T1, a total of 818 subjects were included, and 47 CV events were verified, of which 14 (29.7%) were fatal. For IC, the only factor independently associated with the increased risk of CV events in T1 was the presence of a corneal arch (OR: 9.39; 95% CI: 2.46-35.82). For relatives with positive mutation, factors associated with increased risk of CV events were diabetes mellitus (OR: 7.97; 95% CI: 2.07-30.66) and tobacco consumption (OR: 3.70; 95% CI: 1.09-12.50). In the analysis of the best clinical criteria for the detection of a pathogenic mutation in the IC, the LDL-C values >= 230 mg/dL had the best relationship between sensitivity and specificity. In the ROC curve analysis, the Dutch Lipid Clinic Network (DLCN) score performed better than LDL-C to identify a mutation, the area under the ROC curve was 0.744 (95% CI: 0.704-0.784) and 0.730 (CI 95 %: 0.687-0.774), respectively, p = 0.014. Conclusion: At one year follow-up this cohort identified a high incidence of CV events following entry into a cascade genetic screening program and the predictors of CV events differ between IC and family members. These results may contribute to the development of preventive actions in this group highly susceptible to individuals. In addition, because of the importance of detecting the mutation for a definitive diagnosis of HF and the importance of the cascade being cost effective, the study identified that the single LDL-C criterion >= 230 mg / dl is feasible to indicate IC for the genetic test

A hipercolesterolemia familiar (HF) é uma alteração de origem genética comum que pode se manifestar clinicamente desde o nascimento e provoca um aumento nos níveis plasmáticos de LDL-colesterol (LDL-c), xantomas e doença coronária prematura. Sua detecção e tratamento precoce reduzem a morbidade e mortalidade coronária. A identificação e rastreamento em cascata familiar usando níveis de LDL-c e detecção genética é a estratégia mais aconselhável e rentável para descoberta de novos casos. O tratamento crônico com estatinas reduz o risco cardiovascular da população em geral, contudo, estudos clínicos com estatinas revelam risco cardiovascular residual mesmo após correção das concentrações de LDL-c. Com o surgimento de novas drogas e mais recentemente um inibidor da enzima pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), este estudo enfatizou na investigação específica para aqueles acometidos com defeitos genéticos nessa enzima, por ser de frequência ainda mais rara e pouco estudada, necessitando de melhor investigação na população em estudo a fim de rastrear a ocorrência de mutações patológicas na PCSK9. O objetivo desse estudo foi identificar e caracterizar mutações e/ou deleções patológicas no gene PCSK9 em pacientes com Hipercolesterolemia Familiar provenientes do Hospital das Clínicas de Ribeirão Preto da FMRP/USP selecionados para o teste genético. Foi feito o rastreamento de mutações pelo método Hight Resolution Melting (HRM), de forma prática, rápida e eficiente, onde mutações detectadas foram seqüenciadas. Foram identificadas 7 mutações não patogênicas, caracterizando que a população estudada não apresenta Hipercolesterolemia Familiar associada a mutações no gene PCSK9, fato que não exclui o diagnóstico por outros defeitos genéticas associados a doença. / Familial hypercholesterolemia (FH) is an alteration of common genetic origin that can manifest clinically from birth and which causes an increase in the LDL-cholesterol plasma levels (LDL-c), xanthomas and premature coronary disease. Its early detection and treatment reduce morbidity and coronary mortality. The identification and tracking in familial cascade using levels of LDL-c and genetic detection is the most advisable and profitable strategy to find new cases. The chronic treatment with statins reduces the cardiovascular risk in the population in general. However, clinic studies on statins show a residual cardiovascular risk even after the correction of LDL-c concentrations. With the appearance of new drugs and, more recently, of a proprotein convertase subtilisin/kexin type 9 enzyme inhibitor (PCSK9), this study highlighted the specific investigation for those stricken by genetic defects in this enzyme, once it is even rarer and understudied and needs further investigation in the study\'s population aiming at tracking the occurrence of a pathological mutation in the PCSK9. This study aimed at identifying and characterizing mutations and/or pathological deletions in the PCSK9 gene in patients with Familial Hypercholesterolemia from the RPMS/USP Ribeirão Preto Clinical Hospital which were selected for the genetic test. We performed the mutation tracking by using the High Resolution Melting (HRM) method in a practical, fast and efficient way, where the mutations detected were sequenced. We identified 7 non-pathogenic mutations, showing that the population studied does not present Familial Hypercholesterolemia associated to mutations in the PCSK9 gene, which doesn\'t exclude the diagnosis by other genetic defects associated to the disease.

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Human C-reactive protein (CRP) is the prototypic acute phase protein whose serum concentration increases rapidly during inflammation. CRP is also associated with atherosclerosis; it is deposited at lesion sites where it may interact with modified lipoproteins. There are 2 major questions regarding CRP: 1. How is the serum concentration of CRP regulated? 2. What are the functions of CRP in atherosclerosis? Our first aim was to determine the role of the constitutively expressed transcription factor Oct-1 in regulating CRP gene expression. We found that Oct-1 overexpression inhibited (IL-6+IL-1β)- induced CRP gene expression; maximal inhibition required the binding of Oct-1 to an octamer motif at (-59 to -66) on the CRP promoter. Oct-1 overexpression inhibited both (IL-6+IL-1β)- induced and C/EBPβ-induced CRP gene expression even when the Oct-1 site was deleted. These findings suggest that Oct-1 is a repressor of CRP gene expression that acts via binding to its cognate site on the CRP promoter as well as through indirect interactions with other promoterbound transcription factors. Our second aim was to investigate the interaction of CRP with oxidized low density lipoprotein (ox-LDL). Acidic pH, a hallmark of atherosclerotic lesions, reversibly alters CRP structure and exposes a hidden binding site that enables CRP to bind ox-LDL. Using site-directed mutagenesis we constructed a CRP mutant (E42Q) that showed significant binding to ox-LDL at physiological pH. E42Q CRP required a less acidic pH for maximal binding and bound ox-LDL more efficiently than wild type CRP at any pH. We then examined if reactive oxygen species also induced CRP – ox-LDL interaction. H2O2-treated CRP bound ox-LDL at physiological pH. Like acidic pH, H2O2-treatment induced only a local structural change exposing the ox-LDL binding site. E42Q and H2O2-modified CRP are tools to study the function of CRP in animal models of atherosclerosis, which may not have an inflammatory environment sufficient to modify CRP and induce binding to atherogenic ox-LDL. We conclude that Oct-1 is one of the critical regulators of CRP gene expression, and that CRP can be modified in vitro to convert it into an atherogenic LDL-binding molecule.

<p>Atherosclerosis is a progressive inflammatory disease that is characterized by the accumulation of lipids, infiltrated cells and fibrous elements in large arteries.</p><p>This thesis focuses on the molecular mechanisms behind foam cell formation and inflammation, two central processes in the development of atherosclerosis. More specific, we studied the effects of proinflammatory cytokines on CXCL16 expression and its role as scavenger receptor on macrophages and smooth muscle cells in atherogenesis. CXCL16 is defined as a chemokine and a scavenger receptor, regulating adhesion and chemoattraction of CXCR6 expressing cells and uptake of oxLDL. We show that the expression of CXCL16 and its receptor CXCR6 are more pronounced in human atherosclerotic lesions compared with non-atherosclerotic vessels. Increased expression of CXCL16 was also seen in atherosclerotic aortas of apoE-/- mice compared with aortas of non-atherosclerotic, age-matched C57BL/6 mice. In vitro, IFN gamma induced CXCL16 expression in primary human monocytes and smooth muscle cells which resulted in an increased uptake of oxLDL. Treatment of mice with IFN gamma also induced CXCL16 expression in atherosclerotic lesions. Thus, we have demonstrated a role for IFN gamma in foam cell formation through upregulation of CXCL16. The expression of CXCR6 was defined to the same regions as for CXCL16 in the lesion, indicating the presence of cells able to respond to CXCL16. Consequently, CXCL16 could serve as a molecular link between lipid metabolism and immune activity in atherosclerotic lesion.</p><p>CD137 belongs to the TNF family and mediates several important processes in inflammation. CD137 is involved in the activation of T cells, NK cells, B cells and monocytes and regulate cytokine production, proliferation and apoptosis in these cells. A limited number of studies have demonstrated CD137 expression on smooth muscle cells and endothelial cells. Our results show that CD137 mRNA is higher expressed in human atherosclerotic lesions compared with unaffected vessels. We found that endothelial cells express CD137 in atherosclerotic lesions and that cultured endothelial cells and smooth muscle cells express CD137 and CD137 ligand in vitro. CD137 was regulated differentially by proinflammatory cytokines (i.e. IFN gamma, TNF alpha, IL-1 beta) and bacterial lipopolysaccharide depending on cell type. Furthermore, we investigated the effects of CD137 signalling, demonstrating that binding of the CD137 ligand to its receptor increases proliferation and migration of smooth muscle cells.</p><p>In summary, this thesis has focused on the expression, regulation and role of CXCL16 and CD137, two genes that have not been described earlier in the concept of atherosclerosis. The findings demonstrate some of the molecular mechanisms involved in vascular inflammation and may increase our knowledge about the development of atherosclerosis.</p>

<p>In patients with unstable coronary artery disease (CAD) biochemical markers are emerging as useful tools in clinical management. In this thesis we studied the use of markers of low-density lipoprotein (LDL) oxidation and renal function.</p><p>Our study populations consisted of unstable CAD patients included in the Fast Revascularisation during Instability in Coronary artery disease (FRISC)-II trial and healthy controls. Patients were followed for 2 years regarding death and myocardial infarction (MI).</p><p>Using receiver operating characteristic curve analysis, we found that oxidized low-density lipoprotein (OxLDL), especially when combined with high-density lipoprotein, compared to traditionally measured lipids/lipoproteins, and a new lipoprotein marker, lipoprotein associated-phospholipase A2, was better at discriminating between healthy controls and CAD patients. In patients, OxLDL was found to be an independent prognostic marker associated with an increased risk of MI, of particular use in patients with no evidence of myocardial necrosis. </p><p>In our study on the effects of an early invasive treatment strategy in unstable CAD patients with mild to moderate renal dysfunction (i.e. creatinine clearance <90mL/min) we found that in patients randomized to invasive treatment, the rates of death/MI and MI alone were significantly lower than in patients randomized to non-invasive treatment. In patients treated invasively, no detrimental effects were seen on renal function at follow-up at 6 months. </p><p>In healthy controls, we investigated new markers of renal (cystatin C) and cardio-renal function (N-terminal probrain natriuretic peptide, [NT-proBNP]) regarding reference levels and physiological determinants. We found that cystatin C is influenced by age whereas NT-proBNP is influenced by age and gender.</p><p>Our studies suggest that OxLDL and renal dysfunction are associated with a poor prognosis in unstable CAD patients and that these markers demonstrate potential for clinical use. In the search for new markers related to renal function we have contributed with reference levels of cystatin C and NT-proBNP. </p>

In patients with unstable coronary artery disease (CAD) biochemical markers are emerging as useful tools in clinical management. In this thesis we studied the use of markers of low-density lipoprotein (LDL) oxidation and renal function. Our study populations consisted of unstable CAD patients included in the Fast Revascularisation during Instability in Coronary artery disease (FRISC)-II trial and healthy controls. Patients were followed for 2 years regarding death and myocardial infarction (MI). Using receiver operating characteristic curve analysis, we found that oxidized low-density lipoprotein (OxLDL), especially when combined with high-density lipoprotein, compared to traditionally measured lipids/lipoproteins, and a new lipoprotein marker, lipoprotein associated-phospholipase A2, was better at discriminating between healthy controls and CAD patients. In patients, OxLDL was found to be an independent prognostic marker associated with an increased risk of MI, of particular use in patients with no evidence of myocardial necrosis. In our study on the effects of an early invasive treatment strategy in unstable CAD patients with mild to moderate renal dysfunction (i.e. creatinine clearance &lt;90mL/min) we found that in patients randomized to invasive treatment, the rates of death/MI and MI alone were significantly lower than in patients randomized to non-invasive treatment. In patients treated invasively, no detrimental effects were seen on renal function at follow-up at 6 months. In healthy controls, we investigated new markers of renal (cystatin C) and cardio-renal function (N-terminal probrain natriuretic peptide, [NT-proBNP]) regarding reference levels and physiological determinants. We found that cystatin C is influenced by age whereas NT-proBNP is influenced by age and gender. Our studies suggest that OxLDL and renal dysfunction are associated with a poor prognosis in unstable CAD patients and that these markers demonstrate potential for clinical use. In the search for new markers related to renal function we have contributed with reference levels of cystatin C and NT-proBNP.

Motivation: The prevalence of non-communicable diseases such as diabetes mellitus (DM) and cardiovascular disease (CVD) is rapidly increasing in industrialized societies. Experts believe that lifestyle, and in particular its nutritional aspects, plays a decisive role in increasing the burden of these chronic conditions. Dietary habits would, therefore, be modified to exert a positive impact on the prevention and treatment of chronic diseases of lifestyle. It is believed that the state of hyperglycaemia that is observed following food intake under certain dietary regimes contributes to the development of various metabolic conditions. This is not only true for individuals with poor glycaemic control such as some diabetics, but could also be true for healthy individuals. It would, therefore, be helpful to be able to reduce the amplitude and duration of postprandial hyperglycaemia. Selecting the correct type of carbohydrate (CHO) foods may produce less postprandial hyperglycaemia, representing a possible strategy in the prevention and treatment of chronic metabolic diseases. At the same time, a key focus of sport nutrition is the optimal amount of CHO that an athlete should consume and the optimal timing of consumption. The most important nutritional goals of the athlete are to prepare body CHO stores pre-exercise, provide energy during prolonged exercise and restore glycogen stores during the recovery period. The ultimate aim of these strategies is to maintain CHO availability to the muscle and central nervous system during prolonged moderate to high intensity exercise, since these are important factors in exercise capacity and performance. However, the type of CHO has been studied less often and with less attention to practical concerns than the amount of CHO. The glycaemic index (GI) refers to the blood glucose raising potential of CHO foods and, therefore, influences secretion of insulin. In several metabolic disorders, secretion of insulin is inadequate or impossible, leading to poor glycaemic control. It has been suggested that low GI diets could potentially contribute to a significant improvement of the conditions associated with poor glycaemic control. Insulin secretion is also important to athletes since the rate of glycogen synthesis depends on insulin due to it stimulatory effect on the activity of glycogen synthase. Objectives: Three main objectives were identified for this study. The first was to conduct a meta-analysis of the effects of the GI on markers for CHO and lipid metabolism with the emphasis on randomised controlled trials (RCT's). Secondly, a systematic review was performed to determine the strength of the body of scientific evidence from epidemiological studies combined with RCT's to encourage dieticians to incorporate the GI concept in meal planning. Finally, a systematic review of the effect of the GI in sport performance was conducted on all available literature up to date to investigate whether the application of the GI in an athlete's diet can enhance physical performance. Methodology: For the meta-analysis, the search was for randomised controlled trials with a cross-over or parallel design published in English between 1981 and 2003, investigating the effect of low GI vs high GI diets on markers of carbohydrate and lipid metabolism. The main outcomes were serum fructosamine, glycosylated haemoglobin (HbA1c), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC) and triacylglycerols (TG). For the systematic review, epidemiological studies as well as RCT's investigating the effect of LGI vs HGI diets on markers for carbohydrate and lipid metabolism were used. For the systematic review on the effect of the GI on sport performance, RCT's with either a cross-over or parallel design that were published in English between January 1981 and September 2004 were used. All relevant manuscripts for the systematic reviews as well as meta-analysis were obtained through a literature search on relevant databases such as the Cochrane Central Register of Controlled Trials, MEDLINE (1981 to present), EMBASE, LILACS, SPORTDiscus, ScienceDirect and PubMed. This thesis is presented in the article format. Results and conclusions of the individual manuscripts: For the meta-analysis, literature searches identified 16 studies that met the strict inclusion criteria. Low GI diets significantly reduced fructosamine (p<0.05), HbA1c, (p<0.03), TC(p<0.0001) and tended to reduce LDL-c (p=0.06) compared to high GI diets. No changes were observed in HDL-c and TG concentrations. Results from this meta analysis, therefore, support the use of the GI concept in choosing CHO-containing foods to reduce TC and improve blood glucose control in diabetics. The systematic review combined the results of the preceding meta-analysis and results from epidemiological studies. Prospective epidemiological studies showed improvements in HDL-c concentrations over longer time periods with low GI diets vs. high GI diets, while the RCT's failed to show an improvement in HDL-c over the short-term. This could be attributed to the short intervention period during which the RCT's were conducted. Furthermore, epidemiological studies failed to show positive relationships between LDL-c and TC and low GI diets, while RCT's reported positive results on both these lipids with low GI diets. However, the epidemiological studies, as well as the RCT's showed positive results with low GI diets on markers of CHO metabolism. Taken together, convincing evidence from RCT's as well as epidemiological studies exists to recommend the use of low GI diets to improve markers of CHO as well as of lipid metabolism. 3 From the systematic review regarding the GI and sport performance it does not seem that low GI pre-exercise meals provide any advantages over high GI pre-exercise meals. Although low GI pre-exercise meals may better maintain CHO availability during exercise, low GI pre-exercise meals offer no added advantage over high GI meals regarding performance. Furthermore, the exaggerated metabolic responses from high GI compared to low GI CHO seems not be detrimental to exercise performance. However, athletes who experience hypoglycaemia when consuming CHO-rich feedings in the hour prior to exercise are advised to rather consume low GI pre-exercise meals. No studies have been reported on the GI during exercise. Current evidence suggests a combination of CHO with differing Gl's such as glucose (high GI), sucrose (moderate GI) and fructose (low GI) will deliver the best results in terms of exogenous CHO oxidation due to different transport mechanisms. Although no studies are conducted on the effect of the GI on short-term recovery it is speculated that high GI CHO is most effective when the recovery period is between 0-8 hours, however, evidence suggests that when the recovery period is longer (20-24 hours), the total amount of CHO is more important than the type of CHO. Conclusion: There is an important body of evidence in support of a therapeutic and preventative potential of low GI diets to improve markers for CHO and lipid metabolism. By substituting high GI CHO-rich with low GI CHO-rich foods improved overall metabolic control. In addition, these diets reduced TC, tended to improve LDL-c and might have a positive effect over the long term on HDL-c. This confirms the place for low GI diets in disease prevention and management, particularly in populations characterised by already high incidences of insulin resistance, glucose intolerance and abnormal lipid levels. For athletes it seems that low GI pre-exercise meals do not provide any advantage regarding performance over high GI pre-exercise meals. However, low GI meals can be recommended to athletes who are prone to develop hypoglycaemia after a CHO-rich meal in the hour prior to exercise. No studies have been reported on the effect of the GI during exercise. However, it has been speculated that a combination of CHO with varying Gl's deliver the best results in terms of exogenous CHO oxidation. No studies exist investigating the effect of the GI on short-term recovery, however, it is speculated that high GI CHO-rich foods are suitable when the recovery period is short (0-8 h), while the total amount rather than the type of CHO is important when the recovery period is longer (20-24 h). Therefore, the GI is a scientifically based tool to enable the selection of CHO-containing foods to improve markers for CHO and lipid metabolism as well as to help athletes to prepare optimally for competitions. Recommendations: Although a step nearer has been taken to confirm a place for the GI in human health, additional randomised, controlled, medium and long-term studies as well as more epidemiological studies are needed to investigate further the effect of low GI diets on LDL-c. HDL-c and TG. These studies are essential to investigate the effect of low GI diets on endpoints such as CVD and DM. This will also show whether low GI diets can reduce the risk of diabetic complications such as neuropathy and nephropathy. Furthermore, the public at large must be educated about the usefulness and application of the GI in meal planning. For sport nutrition, randomised controlled trials should be performed to investigate the role of the GI during exercise as well as in sports of longer duration such as cricket and tennis. More studies are needed to elucidate the short-term effect of the GI post-exercise as well as to determine the mechanism of lower glycogen storage with LGI meals post-exercise. / Thesis (Ph.D. (Dietetics))--North-West University, Potchefstroom Campus, 2005.

Proteins modularity enhances the multi-functionality and versatility of proteins by providing such properties as multiple and various ligand-binding sites, increased ligand affinity through the avidity effect, and the juxtaposition of ligand-binding modules near catalytic domains. An NMR-based "dissect-and-build" approach to studying modular protein structure and function has proven very successful, whereby modules are initially characterized individually and then correlated with the overall function of a protein. We have used the dissect-and-build approach and NMR to study two modular protein systems. Chapter 2 details the NMR solution structure of the weak-lysine-binding kringle IV type 8 (KIV8) module from the apolipoprotein(a) (apo(a)) component of lipoprotein(a) was determined and its ligand-binding properties assessed. In vitro studies have demonstrated the importance of the apo(a) KIV7 and KIV8 modules in mediating specific lysine-dependent interactions with the apolipoproteinB-100 (apoB-100) component of LDL in the initial non-covalent step of lipoprotein assembly. Notable differences identified in the lysine binding site (LBS) of the KIV8 were deemed responsible for the differential modes of apoB-100 recognition by KIV7 and KIV8. In addition, the KIV8 structure has brought to light the importance of an RGD sequence at the N-terminus of the apo(a) KIV8 module, which may mediate important apo(a)-integrin interactions. In Chapters 3-6, structure-function studies of the CpGH84C X82 and the CpGH84A dockerin-containing modular pair were conducted to understand how the varying modularity unique to the C-terminal regions of the secreted multi-modular family 84 glycoside hydrolases influences the spreading of Clostridium perfringens. Identification of a CpGH84C cohesin module (X82), and the structural characterization of a dockerin-containing modular pair provides the first evidence for multi-enzyme complex formation mediated by non-cellulosomal cohesin-dockerin interactions. The formation of large hydrolytic enzyme complexes introduces a novel mechanism by which C. perfringens may enhance its role in pathogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-09-27 11:46:38.753