Mark D. P. Willcox

Role of carnitine in disease

Carnitine is a conditionally essential nutrient that plays a vital role in energy production and fatty acid metabolism. Vegetarians possess a greater bioavailability than meat eaters. Distinct deficiencies arise either from genetic mutation of carnitine transporters or in association with other disorders such as liver or kidney disease. Carnitine deficiency occurs in aberrations of carnitine regulation in disorders such as diabetes, sepsis, cardiomyopathy, malnutrition, cirrhosis, endocrine disorders and with aging. Nutritional supplementation of L-carnitine, the biologically active form of carnitine, is ameliorative for uremic patients, and can improve nerve conduction, neuropathic pain and immune function in diabetes patients while it is life-saving for patients suffering primary carnitine deficiency. Clinical application of carnitine holds much promise in a range of neural disorders such as Alzheimers disease, hepatic encephalopathy and other painful neuropathies. Topical application in dry eye offers osmoprotection and modulates immune and inflammatory responses. Carnitine has been recognized as a nutritional supplement in cardiovascular disease and there is increasing evidence that carnitine supplementation may be beneficial in treating obesity, improving glucose intolerance and total energy expenditure.

Complementing genomics with proteomics: The membrane subproteome of Pseudomonas aeruginosa PAO1

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two‐dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane‐spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell‐cell signalling and pathogen‐host interactions in a variety of P. aeruginosa strains.

Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology

Contact lens wear is a successful form of vision correction. However, adverse responses can occur during wear. Many of these adverse responses are produced as a consequence of bacterial colonization of the lens. The present study demonstrated that during asymptomatic contact lens wear lenses are colonized by low levels of bacteria with gram-positive bacteria, such as coagulase negative staphylococci, predominating. Gram-negative bacteria are frequently the causative agents of adverse responses during contact lens wear. Measuring the adhesion of different strains and/or species of bacteria to different contact lens materials demonstrated considerable differences. In particular. Pseudormonas aeruginosa strains Paerl and 6294 and Aeromonas hydrophilia strain Ahyd003 adhered in larger numbers to the highly oxygen permeable contact lenses Balafilcon A compared to hydrogel lenses manufactured from either Etafilcon A or HEMA. Furthermore, after Balafilcon A lenses had been worn for 6 h during the day bacteria were able to adhere in greater numbers to the worn lenses compared to the unworn lenses with increases in adhesion ranging from 243% to 1393%. However, wearing Etafilcon A lenses usually resulted in a decrease in adhesion (22-48%). Bacteria were able to grow after adhesion to lenses soaked in artificial tear fluid and formed biofilms, visualized by scanning confocal microscopy. Chemostat grown bacterial cultures were utilized to enable control of bacterial growth conditions and bacteria were shown to adhere in the greatest numbers if grown under low temperature (25 degrees C compared to 37 degrees C). The changes in growth temperature was shown. using 2D gel electrophoresis, to change the experssion of cell-surface proteins and, using ID gel electrophoresis, to change the expression of surface lipopolysaccharide of P. aeruginosa Paerl. Thus, these surface changes would have been likely to have mediated the increased adhesion to Etafilcon A contact lenses.

A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses.

Aims:  To develop an antimicrobial peptide with broad spectrum activity against bacteria implicated in biomaterial infection of low toxicity to mammalian cells and retaining its antimicrobial activity when covalently bound to a biomaterial surface.

Urinary biomarkers involved in type 2 diabetes: a review

Diabetes mellitus is one of the most challenging health concerns of the 21st century. With at least 30% of the diabetic population remaining undiagnosed, effective and early diagnosis is of critical concern. Development of a diagnostic test, more convenient and reliable than those currently used, would therefore be highly beneficial. Urine as a diagnostic medium allows for non‐invasive detection of biomarkers, including some associated with type 2 diabetes and its complications. This review provides a synopsis of those urinary biomarkers that potentially may provide a basis for the development of improved diagnostic tests. Three main pathways for the sourcing of potential makers are identified: kidney damage, oxidative stress and low‐grade inflammation including atherosclerosis/vascular damage. This review briefly presents each pathway and some of the most relevant urinary biomarkers that may be used to monitor the development or progression of diabetes and its complications. In particular, biomarkers of renal dysfunction such as transferrin, type IV collagen and N‐acetyl‐β‐D‐glucosaminidase might prove to be more sensitive than urinary albumin, the current gold standard, in the detection of incipient nephropathy and risk assessment of cardiovascular disease. Inflammatory markers including orosomucoid, tumour necrosis factor‐α, transforming growth factor‐β, vascular endothelial growth factor and monocyte chemoattractant protein‐1, as well as oxidative stress markers such as 8‐hydroxy‐2′deoxyguanosine may also be useful biomarkers for diagnosis or monitoring of diabetic complications, particularly kidney disease. However, the sensitivity of these markers compared with albumin requires further investigation. Copyright

Elucidation of the antistaphylococcal action of lactoferrin and lysozyme

The cationic tear proteins lactoferrin and lysozyme exhibit co-operative antistaphylococcal properties. The purpose of this study was to determine the mechanism of action of this co-operation on Staphylococcus epidermidis. Following blocking of lipoteichoic acid (LTA) binding sites, the effects on binding of lactoferrin and susceptibility to lactoferrin and lysozyme were determined. The effect of lactoferrin on autolysis and LTA release was also examined. Maximal susceptibility occurred on addition of lactoferrin first followed by lysozyme. Blocking the LTA binding sites both reduced lactoferrin binding and decreased susceptibility. Autolytic activity decreased and LTA release increased in the presence of lactoferrin. These results suggest that binding of lactoferrin to LTA is important in its synergy with lysozyme and interferes with the autolysins present on the LTA. It is proposed that, on binding to the anionic LTA of S. epidermidis, the cationic protein lactoferrin decreases the negative charge, allowing greater accessibility of lysozyme to the underlying peptidoglycan.

Care regimen and lens material influence on silicone hydrogel contact lens deposition

Purpose. To quantitatively detect proteins and cholesterol extracted from worn silicone hydrogel contact lenses and determine the effect of various lens care solutions on deposit accumulation. Methods. Contact lenses, made from different polymers and worn on a daily wear schedule with different lens care solutions, were collected. Lipid and protein deposits were extracted by methanol:chloroform (1:1, v/v) and protein extraction solution (containing urea and surfactant), respectively. Lipid extracts were separated and cholesterol quantified using thin layer chromatography. Protein extracts were quantified using standard techniques. Results. Among all lenses tested, Balafilcon A lenses exhibited greatest extracted cholesterol (4.1 to 8.2 μg/lens) and total protein (5.4 to 23.2 μg/lens). AQuify was the most effective solution in reducing extracted deposits, especially extracted protein, from Balafilcon A lenses. AQuify and Opti-Free RepleniSH solutions were most effective in reducing extracted cholesterol from Senofilcon A and Galyfilcon A lenses, respectively. Use of Opti-Free Express solution resulted in more extracted protein from Lotrafilcon B lenses than use of other solutions. Generally, Lotrafilcon B, Senofilcon A, and Galyfilcon A lenses accumulated relatively low amount of proteins. Lotrafilcon B lenses accumulated the least amount of cholesterol deposit among all lenses tested regardless of solution used. Conclusions. Lens polymer (possibly associated with surface characteristics) is a prominent factor affecting lipid and protein accumulation. Within a lens polymer type, lens care solutions exhibit varying effectiveness in reducing protein and lipid accumulation.

Lipid, lipase and lipocalin differences between tolerant and intolerant contact lens wearers

Purpose. Tear volume is reduced in symptomatic contact lens wearers, evaporation of the ocular tear film may be a cause. In this study we have focussed on symptomatic or intolerant subjects and compared their tear film lipid-related features to those tolerant to soft contact lens wear. Method. Fourteen tolerant and 10 intolerant to lens wear subjects were recruited for this study. Intolerance to lens wear was defined as experiencing dryness symptoms in the first 6 hours of lens wear and consequently not being regular lens wearers. Lipid layer appearance was graded on a 0-5 scale, meibomian gland obstruction was observed, and the McMonnies questionnaire completed. Tears were collected without reflex stimulation. Degraded lipid (tear aldehyde content), secretory phospholipase A2 enzyme (sPLA2) concentration and activity and lipocalin concentration were analysed using spectrophotometry to quantify colour reactions and enzyme linked immunosorbent assays. Statistical results were calculated using non-parametric tests (median ± interquartile range) or chi-squared test. Results. Degradation of polyunsaturated fatty acids and related esters leads to the by-products, malondialdehyde and 4-hydroxy-2(E)-nonenal. Intolerant subjects were found to have significantly (p = 0.004) higher concentrations of these by-products in their tears (0.85 ± 1.0 µM; n = 9) compared to tolerant subjects (0.15 ± 0.15 µM; n = 10). Intolerant subjects (1.86 ± 0.05 ng/µl; n = 9) had significantly more (p = 0.047) sPLA2 enzyme in their tears compared with tolerant subjects (1.80 ± 0.08 ng/µl; n = 12) and significantly more enzyme activity (p = 0.012). Intolerant subjects had significantly higher amounts of lipocalin in their tears (2.40 ± 1.5 µg/µl; n = 10, p < 0.001) compared to tolerant subjects (0.45 µ 0.85 µg/µl; n = 13). Conclusion. Changes to the components of the tear film, however small, can disturb the nature and dynamics of the tear film. Increased lipases, degraded lipids and lipocalins in the aqueous tear film potentiates intolerance to contact lens wear and was associated with increased McMonnies dry eye history scores and symptoms scores.

Contact Lens–Related Adverse Events and the Silicone Hydrogel Lenses and Daily Wear Care System Used

OBJECTIVE To investigate the incidence of adverse events related to the use of varying silicone hydrogel contact lens and lens solution combinations. METHODS Individuals with myopia (N = 558) participated in 1 or more of approximately 40-participant trials in a matrix of 20 silicone hydrogel contact lens and lens-solution combinations. Visits were at baseline, 2 weeks, 1 month, and 3 months. The mean study completion rate was 90% of the expected participant-months (final data set: 840 lens-solution combinations and 2271 participant-months). Adverse events were reported as the first occurrence of each type per 100 participant-months for each lens-solution combination. RESULTS The rate of all corneal infiltrative events (CIEs) was 3.1 per 100 participant-months (range, 0-10.5), and the rate of symptomatic CIEs was 1.7 per 100 participant-months (range, 0-10.5), including 1 case of microbial keratitis (0.04 per 100 participant-months). Rates for CIEs differed substantially among solution groups, with hydrogen peroxide having the lowest rate (0.6 per 100 participant-months; range, 0-0.9). The rate was 0.8 per 100 participant-months (range, 0-8.0) for superior epithelial arcuate lesions, which varied by lens type, 0.04 per 100 participant-months (1 case only) for corneal erosion, and 0.4 per 100 participant-months (range, 0-2.0) for contact lens papillary conjunctivitis, which was modified by type of solution. The rate of solution-induced corneal staining for all lens-solution combinations was 4.7 per 100 participant-months (range, 0-23) and varied significantly based on lens-solution combination (P < .001). CONCLUSIONS The frequency of adverse events varied with silicone hydrogel contact lens and lens solution combinations, with hydrogen peroxide having the lowest incidence of CIEs and solution-induced corneal staining, indicating that lens material and design, type of solution, and solution-lens interactions are likely contributing factors in this mode of lens wear.

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

ABSTRACT The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.