Natural antioxidants and susceptibility of low density lipoproteins to oxidation

In patients with diabetes mellitus, increased oxidative stress may contribute to low density lipoprotein (LDL) oxidation and elevated levels of circulating products of inflammation. A randomised, placebo-controlled study was conducted in patients with type 2 diabetes mellitus to compare the effect of four weeks of supplementation with tomato juice (500 ml/day), alpha-tocopherol (800 IU/day) or vitamin C (500 mg/day) on LDL oxidation, circulating levels of C-reactive protein (C-RP), soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1), and circulating products of lipid peroxidation. Plasma lycopene levels increased nearly three fold with the consumption of tomato juice and LDL resistance (lag time) to copper ion stimulated oxidation increased by 42% (P=0.001). The magnitude of the change with tomato juice was comparable to the corresponding increase during supplementation with a pharmacological dose of alpha-tocopherol (54%, P=0.001). Alpha-tocopherol also decreased plasma C-RP (-49%, P=0.004) and increased plasma cholesterol concentration (9%, P=0.022). Circulating levels of adhesion molecules, erythrocyte TBARS, plasma indices of lipid peroxidation and plasma glucose did not change significantly during the study. These findings indicate drinking tomato juice is an effective way to increase plasma lycopene levels and the intrinsic resistance of LDL to oxidation in diabetic patients. Alpha-tocopherol increases the resistance of LDL to oxidation and reduces systemic inflammatory activity but these potentially anti-atherogenic actions are opposed by a concomitant increase in plasma cholesterol levels. The second study in this thesis focused on the development of a model which may reflect LDL oxidation in the arterial intima more closely than the frequently used copper ion induced oxidation of isolated LDL. Low density lipoproteins can bind to proteoglycans rich in heparin and chondroitin sulphate in the arterial intima and consequently may become a target for atherogenic modification by myeloperoxidase (MPO). Experiments were conducted to examine the susceptibility to peroxidase/hydrogen peroxide (H20 2) catalysed oxidation of resolubilised LDL, that has been precipitated from serum with heparin and from native LDL with heparin, chondroitin sulphate, dextran sulphate, and polyethyleneglycol. In addition, the effects of antioxidants and components of human serum on the oxidation of heparin-LDL in a peroxidase/ H20 2 system were investigated. The LDL from complexes with glycosaminoglycans and dextran sulphate were oxidised rapidly by horse radish peroxidase (HRP) and H202 (mean t1/2max for conjugated diene formation of 3-5 minutes) while there was little oxidation of native LDL and polyethyleneglycol-LDL during the 30 minute incubation period. Aggregated LDL was not oxidised by HRP/H202. The formation of thiobarbituric acid reacting substances (TBARS) paralleled the change in conjugated dienes during oxidation of heparin-LDL. Heparin-LDL was also more rapidly oxidised than native LDL by MPO/H202. Oxidation of heparin-LDL by peroxidases did not require free tyrosine and was almost totally inhibited by butylated hydroxytoluene (BHT) and ascorbate, and was unaffected by vitamin E or urate. Increasing concentrations (0-14.9%) of betalipoprotein deficient serum (BLPDS) significantly (P<0.0001) inhibited the formation of TBARS during heparin-LDL oxidation catalysed by HRP and MPO. The inhibitory activity was removed by dialysis and gel-filtration of BLPDS and was not restored by addition of physiological levels of ascorbate, tyrosine and reduced thiols (cysteine) to gel-filtered BLPDS. These results indicate that LDL can form complexes with glycosaminoglycans rendering them particularly susceptible to oxidation by peroxidases. Furthermore LDL oxidation may be inhibited by small, water soluble compounds in the human serum but not by vitamin E. These findings may be relevant to the formation of oxidatively modified LDL in the artery wall

Anti-diabetic effect of a preparation of vitamins, minerals and trace elements in diabetic rats: a gender difference

BACKGROUND: Although multivitamin products are widely used as dietary supplements to maintain health or as special medical food in certain diseases, the effects of these products were not investigated in diabetes mellitus, a major cardiovascular risk factor. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) for human use affects the severity of experimental diabetes. METHODS: Two days old neonatal Wistar rats from both genders were injected with 100 mg/kg of streptozotocin or its vehicle to induce diabetes. At week 4, rats were fed with an MVT preparation or vehicle for 8 weeks. Well established diagnostic parameters of diabetes, i.e. fasting blood glucose and oral glucose tolerance test were performed at week 4, 8 and 12. Moreover, serum insulin and blood HbA1c were measured at week 12. RESULTS: An impaired glucose tolerance has been found in streptozotocin-treated rats in both genders at week 4. In males, fasting blood glucose and HbA1c were significantly increased and glucose tolerance and serum insulin was decreased at week 12 in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. All of the diagnostic parameters of diabetes were significantly improved by MVT treatment in male rats. In females, streptozotocin treatment resulted in a less severe prediabetic-like phenotype as only glucose tolerance and HbA1c were altered by the end of the study in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. MVT treatment failed to improve the diagnostic parameters of diabetes in female streptozotocin-treated rats. CONCLUSION: This is the first demonstration that MVT significantly attenuates the progression of diabetes in male rats with chronic experimental diabetes. Moreover, we have confirmed that females are less sensitive to STZ-induced diabetes and MVT preparation did not show protection against prediabetic state. This may suggest a gender difference in the pathogenesis of diabetes

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples.

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose.fals

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose

The detrimental impact of extracellular bacterial proteases on wound healing

In addition to clinical signs of infection (e.g. inflammation, purulence and pain), a microbial count of ≥105 colony‐forming units/g has historically been used to define wound infection. However, it is increasingly recognised that, rather than a high bioburden level alone being detrimental to wound healing, it is the virulence of the invading microorganism and the host's immune status that can affect clinical outcomes. Bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, have developed a range of virulence factors to help them overcome host defences and proliferate within the underlying soft tissue. More specifically, bacterial proteases are one such virulence factor that has been implicated in promoting the invasion and destruction of the host tissue. Because of the complexities of microorganisms, the proteases can negatively impact the wound environment, leading to delayed wound healing. The aim of the present paper is to describe various extracellular bacterial proteases; review the impact they have on the wound environment, the host immune response and biofilms; and discuss potential wound management strategies against them. The evidence discussed suggests that proteases may play a profound role in wound infections, contribute to the development of an inflammatory response and impede wound healing