Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

This study compared the molecular lipidomic profi le of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identifi ed 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profi le in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N -acyltaurines were signifi cantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were signifi cantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identifi ed individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These fi ndings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profi le of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease. -Reis, A., A. Rudnitskaya, P. Chariyavilaskul, N. Dhaun, V. Melville, J. Goddard, D. J. Webb, A. R. Pitt, and C. M. Spickett. Topdown lipidomics of low density lipoprotein reveal altered lipid profi les in advanced chronic kidney disease. J. Lipid Res. 2015.

Relationship between the microcirculation and the pathological changes in the spradic and variant forms of Creutzfeldt-Jakob Disease (CJD)

This chapter is concerned with the influence of the brain microcirculation on the development of the pathological changes in Creutzfeldt-Jakob disease (CJD). Hence, the spatial correlations between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles were studied in immunolabelled sections of the cerebral cortex, hippocampus, and cerebellum in two subtypes of CJD, viz., sporadic Creutzfeldt-Jakob disease (sCJD) and variant CJD (vCJD). In sCJD, both the vacuolation and the ‘synaptic-type’ PrP deposits were spatially correlated with the blood vessels; the PrP deposits being the more strongly correlated than the vacuoles. In vCJD, there were no significant spatial correlations between either the vacuolation or the diffuse-type of PrP deposit and the microvessels. By contrast, a consistent pattern of spatial correlation was observed in gyri of the cerebral cortex between the florid PrP deposits and blood vessels. In both sCJD and vCJD, the frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex and in the upper compared with the lower laminae. In conclusion, the microcirculation appears to be more significantly involved in determining the pathological changes in sCJD than in vCJD. The spatial correlations of the florid PrP deposits in vCJD and the synaptic deposits in sCJD and the blood vessels may be attributable to factors associated with the microcirculation which enhance the aggregation of PrP molecules rather than representing a possible haematogenous spread of the disease.

Spatial correlations between beta-amyloid (Abeta) deposits and blood vessels in early-onset Alzheimer's disease

In cases of late-onset Alzheimer’s disease (AD), there is a spatial correlation between the classsic ‘cored’ type of Beta-amyloid (Abeta) deposit and the large vertically penetrating arterioles in the cerebral cortex suggesting that blood vessels are involved in the pathogenesis of the classic deposits. In this chapter, the spatial correlations between the diffuse, primitive, and classic Abeta deposits and blood vessels were studied in 10 cases of early-onset AD in the age range 40 – 65 years. Sections of frontal cortex were immunostained with antibodies against Abeta?and with collagen IV to reveal the Abeta deposits and blood vessel profiles. In the early-onset cases as a whole, all types of Abeta? deposit and blood vessel profiles were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Abeta deposits and the larger (>10µm) and smaller diameter (<10?m) blood vessel profiles in one and three cases respectively. The primitive and classic Abeta deposits were spatially correlated with larger and smaller blood vessels both in three and four cases respectively. Spatial correlations between the Abeta deposits and blood vessels may be more prevalent in cases expressing amyloid precursor protein (APP) than presenilin 1 (PSEN1) mutations. Apolipoprotein E (Apo E) genotype of the patient did not appear to influence the spatial correlation with blood vessel profiles. The data suggest that the larger diameter blood vessels are less important in the pathogenesis of the classic Abeta deposits in early-onset compared with late-onset AD.

Description and critical appraisal of principal components analysis (PCA) methodology applied to pulsed-field gel electrophoresis profiles of methicillin-resistant Staphylococcus aureus isolates

Principal components analysis (PCA) has been described for over 50 years; however, it is rarely applied to the analysis of epidemiological data. In this study PCA was critically appraised in its ability to reveal relationships between pulsed-field gel electrophoresis (PFGE) profiles of methicillin- resistant Staphylococcus aureus (MRSA) in comparison to the more commonly employed cluster analysis and representation by dendrograms. The PFGE type following SmaI chromosomal digest was determined for 44 multidrug-resistant hospital-acquired methicillin-resistant S. aureus (MR-HA-MRSA) isolates, two multidrug-resistant community-acquired MRSA (MR-CA-MRSA), 50 hospital-acquired MRSA (HA-MRSA) isolates (from the University Hospital Birmingham, NHS Trust, UK) and 34 community-acquired MRSA (CA-MRSA) isolates (from general practitioners in Birmingham, UK). Strain relatedness was determined using Dice band-matching with UPGMA clustering and PCA. The results indicated that PCA revealed relationships between MRSA strains, which were more strongly correlated with known epidemiology, most likely because, unlike cluster analysis, PCA does not have the constraint of generating a hierarchic classification. In addition, PCA provides the opportunity for further analysis to identify key polymorphic bands within complex genotypic profiles, which is not always possible with dendrograms. Here we provide a detailed description of a PCA method for the analysis of PFGE profiles to complement further the epidemiological study of infectious disease. © 2005 Elsevier B.V. All rights reserved.

Ocular and systemic markers for vascular function in those at risk of type 2 diabetes mellitus and cardiovascular disease

The devastating impact of Type 2 Diabetes Mellitus (T2DM) -related morbidity and mortality on global healthcare is escalating with higher prevalences of obesity, poor diet, and sedentary lifestyles. Therefore, the clinical need for early diagnosis and prevention in groups of high-risk individuals is necessary. The purpose of this thesis was to investigate the use of surrogate markers, namely retinal vascular function, to determine future vascular endothelial dysfunction, atherosclerosis, large vessel disease and cardiovascular risk in certain groups. This namely covered normoglycaemic and normotensive South Asians (SAs), those with Impaired-Glucose Tolerance (IGT) and individuals with a familial history (FH) of T2DM. Additionally the effect of overweight and obesity was studied. The techniques and modified protocols adopted for this thesis involved the investigation of endothelial function by means of vascular reactivity at the ocular and systemic level. Furthermore, the relationships between retinal and systemic function with circulating markers for endothelial cell function and cardiovascular risk markers were explored. The principal studies and findings of the research were: Vascular Function in Normoglycaemic Individuals with and without a FH of T2DM WE FH individuals exhibited higher levels of total cholesterol levels that correlated well with the retinal arterial dilation amplitude to flicker light stimulus. However this did not extend to noticeable differences in markers for endothelial cell damage and impaired retinal and systemic function. Vascular Function in Normoglycaemic South-Asians vs. White-Europeans without a FH and Vascular Disturbances Compared to healthy WEs (normo -glycaemic and -tensive), SA participants exhibited levels of dyslipidaemia and a state of oxidative stress that extended to impaired vascular function as detected by reduced brachial artery flow-mediated dilation, slower retinal arterial vessel dilation reaction times (Appendix 3) and steeper constriction profiles. Furthermore, gender sub-group analysis presented in a sub-chapter shows that SA males demonstrated 24-hour systemic blood pressure (BP) and heart rate variability (HRV) abnormalities and heightened cardiovascular disease (CVD) risk. Vascular Function in Individuals Newly Diagnosed with IGT as compared to Normoglycaemic Healthy Controls Newly-diagnosed WE and SA IGT patients showed a greater risk for CVD and T2DM progression by means of 24-hour BP abnormalities, dyslipidaemia, increased carotid artery intimal-media thickness (c-IMT), Framingham scores and cholesterol ratios. Additionally, pre-clinical markers for oxidative stress and endothelial dysfunction, as evident by significantly lower levels of plasma glutathione and increased levels of von-Willebrand factor in IGT individuals, extended to impaired vascular systemic and retinal function compared to normal controls. This originally shows retinal, systemic and biochemical disturbances in newly-diagnosed IGT not previously reported before. Vascular Function in Normal, Overweight and Obese Individuals of SA and WE Ethnicity In addition to the intended study chapters, the thesis also investigated the influence of obesity and overweight on vascular function. Most importantly, it was found for the first time that compared to lean individuals it was overweight and not obese individuals that exhibited signs of vascular systemic and ocular dysfunction that was evident alongside markers of atherosclerosis, CVD risk and endothelial damage.

Spatial correlations between beta-amyloid (A beta) deposits and blood vessels in familial Alzheimer's disease

In sporadic Alzheimer’s disease (SAD), the classic (‘dense-cored’) ß-amyloid (Aß) deposits are aggregated around the larger blood vessels in the upper laminae of the cerebral cortex. To determine whether a similar relationship exists in familial AD (FAD), the spatial correlations between the diffuse, primitive, and classic ß-amyloid (Aß deposits and blood vessels were studied in ten FAD cases including cases linked to amyloid precursor protein (APP) and presenilin (PSEN) gene mutations and expressing apolipoprotein E (apo E) allele E4. Sections of frontal cortex were immunolabelled with antibodies against Aß and with collagen IV to reveal the Aß deposits and blood vessel profiles. In the FAD cases as a whole, Aßdeposits were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Aßdeposits and the larger (>10 µm) and smaller diameter (<10 µm) blood vessels in one and three cases respectively. The primitive Aß deposits were spatially correlated with larger and smaller blood vessels each in four cases and the classic deposits in three and four cases respectively. Apo E genotype of the patient did not influence spatial correlation with blood vessels. Hence, spatial correlations between the classic deposits and larger diameter blood vessels were significantly less frequent in FAD compared with SAD. It was concluded that both Aß deposit morphology and AD subtype determine spatial correlations with blood vessels in AD.

Classic beta-amyloid deposits cluster around large diameter blood vessels rather than capillaries in sporadic Alzheimer's disease

Various hypotheses could explain the relationship between beta-amyloid (Abeta) deposition and the vasculature in Alzheimer's disease (AD). Amyloid deposition may reduce capillary density, affect endothelial cells of blood vessels, result in diffusion from blood vessels, or interfere with the perivascular clearance mechanism. Hence, the spatial pattern of the classic ('cored') type of Abeta deposit was studied in the upper laminae (I,II/III) of the superior frontal gyrus in nine cases of sporadic AD (SAD). Sections were immunostained with antibodies against Abeta and with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a positive spatial correlation between the clusters of the classic deposits and the larger diameter (>10 microm) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 microm) capillaries. There were no negative correlations between the density of Abeta deposits and the smaller diameter capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessel profiles. In addition, the density of the classic deposits declined as a negative exponential function with distance from a vertically penetrating arteriole. These results suggest that the classic Abeta deposits cluster around the larger blood vessels in the upper laminae of the frontal cortex. This aggregation could result from diffusion of proteins from blood vessels or from overloading the system of perivascular clearance from the brain.

Does the neuropathology of human patients with variant Creutzfeldt-Jakob disease reflect haematogenous spread of the disease?

To test the hypothesis that the distribution of the pathology in variant Creutzfeldt-Jakob disease (vCJD) represents haematogenous spread of the disease, we studied the spatial correlation between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles in the cerebral cortex, hippocampus, dentate gyrus, and cerebellum of 11 cases of the disease. In the majority of areas, there were no significant spatial correlations between either the vacuolation or the diffuse type of PrP deposit and the blood vessels. By contrast, a consistent pattern of spatial correlation was observed between the florid PrP deposits and blood vessels mainly in the cerebral cortex. The frequency of positive spatial correlations was similar in different anatomical areas of the cerebral cortex and in the upper compared with the lower laminae. Hence, with the exception of the florid deposits, the data do not demonstrate a spatial relationship between the pathological features of vCJD and blood vessels. The spatial correlation of the florid deposits and blood vessels may be attributable to factors associated with the blood vessels that promote the aggregation of PrP to form a condensed core rather than reflecting the haematogenous spread of the disease. © 2003 Elsevier Ireland Ltd. All rights reserved.

Florid prion protein (PrP) plaques in patients with variant Creutzfeldt-Jakob disease (vCJD) are spatially related to blood vessels

This study tested the hypothesis that variations in the density of the florid prion protein (PrP) plaques in the brain of patients with variant Creutzfeldt-Jakob disease (vCJD) were spatially related to blood vessels. In 81% of areas of the cerebral cortex sampled and in 37% of the remaining areas, which included the hippocampus, dentate gyrus, and cerebellum, there was a positive spatial correlation between the density of the florid plaques and the larger blood vessel profiles. The frequency of the positive spatial correlations was similar in different anatomical areas of the cerebral cortex and in the upper compared with the lower cortical laminae. The data support the hypothesis that the florid plaques cluster around the larger blood vessels in vCJD, the density of associated plaques increasing with vessel size. The development of florid plaques close to blood vessels may be due to factors associated with the blood vessels that enhance the aggregation of PrP to form the dense cores of florid plaques and is unlikely to reflect the haematogenous spread of PrP into the brain.

Pathological changes in the sporadic and variant forms Of Creutzfeldt-Jakob Disease (CJD) are spatially related to blood vessels

The objective of this article was to determine whether the pathological changes of Creutzfeldt-Jacob disease (CJD) were related to the brain microcirculation. Hence, the spatial correlations between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles were studied in immunolabelled sections of the cerebral cortex, hippocampus, and cerebellum in two subtypes of CJD, viz., sporadic CJD (sCJD) and variant CJD (vCJD). In sCJD, both the vacuolation and the ‘synaptic-type’ PrP deposits were spatially correlated with the microvessels; the PrP deposits being more strongly correlated than the vacuoles. In vCJD, there were no significant spatial correlations between either the vacuolation or the diffuse-type of PrP deposit and the microvessels. By contrast, a consistent pattern of spatial correlation was observed in gyri of the cerebral cortex between the florid PrP deposits and microvessels. In both sCJD and vCJD, the frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex and in the upper compared with the lower laminae. In conclusion, the microcirculation may be more significantly involved in determining the pathological changes in sCJD than in vCJD. The spatial correlations of the florid PrP deposits in vCJD and the synaptic deposits in sCJD and the blood vessels may be attributable to factors associated with the microcirculation which enhance the aggregation of PrP molecules rather than representing a possible haematogenous spread of the disease. S

Dispersion of classic beta-amyloid deposits around blood vessels in Alzheimer’s disease

The spatial pattern of the classic (‘cored’) type of beta-amyloid (Abeta) deposit was studied in the upper laminae of the superior temporal gyrus in 9 cases of sporadic Alzheimer’s disease (SAD). Abeta stained tissue was counterstained with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a spatial correlation between the clusters of the classic deposits and the larger diameter (>10 micron) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 micron) capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessels. In addition, the density of the classic deposits declined as a negative exponential function with distance from the vertically penetrating arterioles. These results suggest that the classic Abeta deposits cluster around the larger blood vessels in the frontal cortex and that diffusion of proteins from these blood vessels could be involved in the pathogenesis of the classic deposits in SAD.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Dietary enrichment by almond supplementation: effects on risk factors for cardiovascular disease

Cardiovascular disease (CVD) is the leading cause of death in Europe responsible for more than 4.3 million deaths annually. The World Health Organisation funded the Monica project (1980s-1990s) which monitored ten million subjects aged 22-6Syrs, and demonstrated that coronary heart disease (CHD) mortality declined over 10 years, was due in two thirds of cases to reduced incidence of CHD (reduced risk behaviours e.g. poor diet and smoking) and one third by improved treatments. Epidemiological evidence suggests diets rich in antioxidants decrease incidence of CVD. Regular consumption of nuts, rich in vitamin E and polyphenols reduces atherosclerosis, an important risk for heart disease. Intervention studies to date using alpha tocopherol (an active component of vitamin E) have not consistently proved beneficial. This thesis aims to investigate the effect of almond supplementation on vascular risk factors in healthy young males (18-3Syrs); mature males and female(>SOyrs); and males considered at increased risk of CVD (18-3Syrs) in a cohort of 67 subjects. The effects of almond intake were assessed after 2Sg/d for four weeks followed by SOg/d for four weeks and compared to a control group which did not consume almonds or change their diet. Cardiovascular risk was assessed by plasma lipid profiles, apolipoprotein A1, plasma nitrates/nitrates, vascular flow, BMl, blood pressure, sVCAM-1 and protein oxidation. Systolic and diastolic blood pressures were reduced in almond supplemented volunteers but not in controls. Dietary monounsaturated fatty acids, polyunsaturated fatty acid content and total dietary fats were increased by almond supplementation. Neither sVCAM-1, venous occlusion plethysmography nor plasma nitrite levels were affected by almond intake in any independent group. No significant changes in plasma lipids, and apolipoprotein A1 were observed. In conclusion almonds supplementation caused a reduction in blood pressure that may be due to increased sensitivity of the baroreceptors after increased monounsaturated fatty acid intake.

Pathological changes in the sporadic and variant forms of Creutzfeldt-Jakob Disease (CJD) are spatially related to blood vessels

The objective of this article was to determine whether the pathological changes of Creutzfeldt-Jacob disease (CJD) were related to the brain microcirculation. Hence, the spatial correlations between the vacuolation, prion protein (PrP) deposits, and the blood vessel profiles were studied in immunolabelled sections of the cerebral cortex, hippocampus, and cerebellum in two subtypes of CJD, viz., sporadic CJD (sCJD) and variant CJD (vCJD). In sCJD, both the vacuolation and the ‘synaptic-type’ PrP deposits were spatially correlated with the microvessels; the PrP deposits being more strongly correlated than the vacuoles. In vCJD, there were no significant spatial correlations between either the vacuolation or the diffuse-type of PrP deposit and the microvessels. By contrast, a consistent pattern of spatial correlation was observed in gyri of the cerebral cortex between the florid PrP deposits and microvessels. In both sCJD and vCJD, the frequency of positive spatial correlations was similar in the different gyri of the cerebral cortex and in the upper compared with the lower laminae. In conclusion, the microcirculation may be more significantly involved in determining the pathological changes in sCJD than in vCJD. The spatial correlations of the florid PrP deposits in vCJD and the synaptic deposits in sCJD and the blood vessels may be attributable to factors associated with the microcirculation which enhance the aggregation of PrP molecules rather than representing a possible haematogenous spread of the disease.

A quantitative analysis of optic nerve axons in elderly control subjects and patients with Alzheimer's disease

Objective: To study the density and cross-sectional area of axons in the optic nerve in elderly control subjects and in cases of Alzheimer's disease (AD) using an image analysis system. Methods: Sections of optic nerves from control and AD patients were stained with toluidine blue to reveal axon profiles. Results: The density of axons was reduced in both the center and peripheral portions of the optic nerve in AD compared with control patients. Analysis of axons with different cross-sectional areas suggested a specific loss of the smaller sized axons in AD, i.e., those with areas less that 1.99 μm2. An analysis of axons >11 μm2 in cross-sectional area suggested no specific loss of the larger axons in this group of patients. Conclusions: The data suggest that image analysis provides an accurate and reproducible method of quantifying axons in the optic nerve. In addition, the data suggest that axons are lost throughout the optic nerve with a specific loss of the smaller-sized axons. Loss of the smaller axons may explain the deficits in color vision observed in a significant proportion of patients with AD.