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Biomedical subjects

M D Willcox

Publications and source records attributed to M D Willcox.

At least 73 records · Page 4Linked to original sources
PubMed1997

Detection and specificity of anti-Staphylococcus intermedius secretory IgA in human tears.

PURPOSE: Secretory IgA (sIgA) is the predominant immunoglobulin present in tears that protects the ocular surface against various antigens. Staphylococcus intermedius is a member of the normal ocular microbiota. METHODS: The presence and the specificity of immunoglobulin A to S. intermedius was determined by fluorescent assay, ELISA and western blots. RESULTS: Three immunodominant antigens of S. intermedius were detected of 145, 127 and 61 kDa. Staphylococcus intermedius-specific IgA cross reacted with Staphylococcus aureus but not with Gram negative bacteria. CONCLUSIONS: This indicates that specific IgA may play an important role in the protection of the eye by limiting the levels of Gram positive normal microbiota and defending against the more pathogenic S. aureus.

Antibodies, Bacterial↗
PubMed1997

Interactions between the constitutive host defences of tears and Staphylococcus epidermidis.

As part of the normal microbiota of the external eye, Staphylococcus epidermidis has probably developed strategies to overcome tear defences. The interaction of this bacteria with constitutive tear proteins was examined. Isolates of S. epidermidis grown in 10% serum or artificial tear fluid containing lysozyme or lactoferrin were resistant to the action of these proteins. Using ELISA, cell wall binding of C3, vitronectin and lactoferrin differed quantitatively between strains and in closed-eye compared to open-eye conditions. No differences were observed between ocular and non-ocular strains. This suggests that ocular isolates originate from the general host microbiota and S. epidermidis isolates are resistant to individual constitutive tear proteins.

Bacterial Adhesion↗
PubMed1997

Growth of gram-negative bacteria in a simulated ocular environment.

PURPOSE: Gram-negative bacteria are major pathogens in the ocular environment, especially when contact lenses are worn. The source of many of these organisms, including Pseudomonas aeruginosa, is thought to be environmental. METHODS: A defined medium is described here that resembles both tear fluid and the environment they are derived from. RESULTS: Gram-negative ocular bacteria were found to grow successfully in this medium. Bactericidal tear proteins were added to this medium and pathogenic strains were not only able to resist their effects but were able to use them for growth. CONCLUSION: Utilization of tear proteins for growth may be an important virulence factor of pathogenic bacteria in the ocular environment.

Eye Proteins↗
PubMed1997

Presence of inflammatory mediators in the tears of contact lens wearers and non-contact lens wearers.

The present studies have demonstrated the levels of N-methyl histamine in tears from normal physiological states, pathological states and contact lens wear. Histamine was significantly increased in closed eye tears compared with both open and reflex tears. Tears from other ocular conditions had low levels of histamine except for environmental hypersensitivity, which contained significantly elevated levels compared to normal tear types. Interestingly, tears from asymptomatic contact lens wearers had significant levels of histamine whereas tears from contact lens adverse events had lower levels, possibly reflecting a change in histamine metabolism.

Analysis of Variance↗
PubMed1997

Role of tear fluid in the growth of gram-negative bacteria on contact lenses.

Incidents of CLARE (contact lens induced acute red eye) are associated with high numbers of Gram-negative bacteria on the lens surface. This study found that most strains of bacteria implicated in CLARE adhere poorly to the lens surface regardless of whether the lens surface was clean or had been worn in the eye. The high numbers of bacteria on the lens surface result from bacterial growth on the lens surface after adhesion has occurred. Using an artificial tear formulation and real tears, this study demonstrated that the tear fluid provides the necessary nutrients required for bacterial growth.

Bacterial Adhesion↗
PubMed1997

Ocular microbiota and polymorphonuclear leucocyte recruitment during overnight contact lens wear.

Bacterial colonization of the ocular surface and contact lens (CL) and recruitment of polymorphonuclear leucocytes (PMN) during overnight CL wear was examined in 11 asymptomatic wearers. The ocular surface was more frequently colonized than the CL, with commensal bacteria (P < 0.05). Following sleep, more bacteria were recovered from the CL compared with daily use (P < 0.05), and fewer PMN were recruited compared to sleep without CL wear (P < 0.05). Overnight CL wear may inhibit physiological PMN recruitment to the cornea by preventing their access, by modifying the chemotactic signal or by altering the activation state of the recruited cells.

Bacteria↗
PubMed1997

Effect of hydrogel lens wear on the major tear proteins during extended wear.

PURPOSE: The purpose of the present study was to determine the whether contact lens wear disturbed the levels of tear proteins and to further determine whether this was a transient or continuous disruption. METHODS: Lactoferrin, lysozyme and albumin were quantitated from tears of neophyte patients and were compared with the levels of these proteins in contact lens wearers after one and six nights and 6 months of extended wear. The quantitation of these tear proteins was performed by sandwich ELISA and turbidimetric assay. RESULTS: Results showed that there were no statistically significant changes in the concentration of any of the proteins investigated. CONCLUSIONS: Extended wear of hydrogel lenses does not appear to alter the concentration of the major tear film proteins, indicating that the tear film is constantly replenished to maintain protein levels, which are depleted due to protein adsorption to the lens surface.

Albumins↗
PubMed1997

Adhesion and growth of Serratia marcescens on artificial closed eye tears soaked hydrogel contact lenses.

The adhesion to hydrogel contact lenses and growth of Serratia marcescens on artificial tear fluid (ATF) soaked lenses was investigated. Results indicated that a corneal ulcer isolate adhered more avidly to lenses; ATF increased adhesion for all strains tested. The contact lens induced acute red eye (CLARE) isolate adhered poorly, however, it grew to a larger extent on ATF-coated lenses. The ability of the corneal ulcer isolate to adhere to lenses may be an important factor in its pathogenicity whereas the ability of the CLARE isolate to grow on the lens in the presence of antimicrobial tear proteins may be important in the development of inflammation.

Bacterial Adhesion↗
PubMed1997

Modulation of cytokine production from an EpiOcular corneal cell culture model in response to Staphylococcus aureus superantigen.

The present study investigated the hypothesis that Staphylococcus aureus enterotoxin B (SEB) produces epithelial cell death and releases inflammatory cytokines that produce stromal infiltration during contact lens induced peripheral ulceration. Epithelial cells were incubated with different doses of SEB for various time periods. Culture supernatants were assayed for cytokines IL-1 beta, IL-6 and chemotactic agents IL-8 and LTB4 SEB induced the production of IL-1 beta and IL-8. Epithelial cells exposed for longer periods (48 h) with low concentrations of SEB produced significantly higher levels (P < 0.02) of IL-1 beta and IL-8 (P < 0.05) compared to a 24 h exposure. SEB did not induce the production of IL-6 and LTB4.

Cell Death↗
PubMed1997

Complement and complement regulatory proteins in human tears.

PURPOSE: The complement system is part of the innate defense system of the body, and it contributes to inflammatory conditions. The current study examined tears for the presence of complement components, the activity of the components, and the presence of regulatory components. METHODS: The significance of a functional complement system in tears was examined in four ways. First, the presence and concentration of complement components in tear samples (open-eye, closed-eye, and reflex tears) was examined by sandwich enzyme-linked immunosorbent assay. Second, the presence of an active pathway in each tear type was established by supplementation of complement-deficient sera. Third, Western blotting of tear samples was used to determine whether complement components were activated in tears. Fourth, the presence of regulatory components was examined by enzyme-linked immunosorbent assay and by the inhibition of the ability of tears to supplement deficient sera. RESULTS: Components C1q, C3, factor B, C4, C5, and C9 were detected in closed-eye tears. Only C3, factor B, and C4 were detected in open-eye and reflex tears. Tears were able to supplement complement-deficient sera, indicating that the components were in an active state. Complement components C3, factor B, C4, and C9 were activated in closed-eye tears. The regulatory protein decay-accelerating factor was found only in closed-eye tears. Lactoferrin, another regulatory protein present in all tear types, was shown to inhibit complement-mediated red blood cell lysis, although the inhibition by closed-eye tear lactoferrin was reduced compared to that isolated from other tear types. CONCLUSIONS: This study has demonstrated that the complement system in tears was functionally active and that the concentration of all components was increased greatly in closed-eye tears. In spite of the presence of regulatory proteins, proteins of the complement cascade in tears were shown to be activated.

Blinking↗
PubMed1997

Effect of different diets on oral bacteria and caries activity in Sprague-Dawley rats.

The conventional rat model of dental caries has been used extensively to measure the contribution of various inoculated bacteria or diets to dental caries production. Only limited information is available on the effect of different diets on the oral bacterial community as a whole. In the present study, using selective and non-selective media, the oral bacterial populations of 21-day-old Sprague-Dawley rats fed diets with commercial rat and mouse cubes (control), high-starch (56%) and sucrose (56%) diets for 21 days were investigated. The high-starch diet did not significantly change the proportion of oral bacteria compared with the control group; the high-sucrose diet significantly enhanced the total cultivable bacteria, particularly Actinomyces naeslundii, Streptococcus rattus and Streptococcus suis-like bacteria. The order of diets with respect to the production of dental caries was sucrose > starch > normal. Thus the high-sucrose diet changed the oral bacterial population of Sprague-Dawley rats and these changes were reflected in the increased level of dental caries.

Actinomyces↗
PubMed1996

An examination of the clonal variants of Serratia marcescens that infect the eye during contact lens wear.

Serratia marcescens colonises contact lenses during wear, although the frequency of isolation is generally low (0.6% contamination rate). A method for typing the S. marcescens colonising the eye or contact lens was developed, based upon ribotyping, serotyping and biotyping. Twelve different types of S. marcescens were isolated from the eyes, contact lenses, contact lens cases and fingers of contact lens wearers in the Sydney area over a 2-year period. There was no evidence of a specific type being more readily able to colonise the contact lenses than other types. Indeed, eight S. marcescens strains were isolated from the lenses and these belonged to seven types. The diversity of types isolated from the eye indicates that there is probably not a subset of S. marcescens that can colonise the eye, although the results suggest that the types of strains isolated from contact lenses are different from those isolated from nosocomial infections.

Bacterial Typing Techniques↗
PubMed1996

Identification and classification of species within the Streptococcus sanguis group.

A biochemical identification scheme has been produced that allows for the differentiation of nine distinct species within the Streptococcus sanguis group. The species are S. sanguis, S. gordonii, S. crista, S. oralis, S. parasanguis, S. defectivus, S. adjacens and S. mitis, which have been previously described, and a new species, S. australis; in addition two distinct subspecies, S. oralis subsp. corona and S. oralis subsp. mitior, have been identified. DNA-DNA hybridization confirmed the separation of strains into the species. A new type of peptidoglycan peptide linkage, lys-ala-gly was also found within the species S. parasanguis and S. australis that has not been observed within the streptococci previously. DNA fingerprinting was shown to be a useful method for discriminating between strains within species, but did not allow discrimination between species.

Alanine↗
PubMed1996

Haemophilus influenzae adherent to contact lenses associated with production of acute ocular inflammation.

Ten episodes of adverse responses to contact lens wear, including contact lens-induced acute red eye (CLARE), in which Haemophilus influenzae was isolated from contact lenses and/or from one of the external ocular sites at the time of the event, are described. All episodes occurred in patients wearing disposable hydrogel lenses on a 6-night extended-wear schedule. Two of the patients had recurrent episodes. H. influenzae was usually isolated in large numbers, and other bacteria or fungi colonizing the contact lens or the external ocular surface were usually present in low numbers. Those patients who were colonized with H. influenzae were more than 100 times as likely to have had a CLARE or infiltrative response than those subjects who were not colonized with this bacterium. H. influenzae colonization of the contact lens and eye may be subsequent to colonization of the nasopharynx because four of the seven patients presented with fever at the time of the event, with concurrent upper respiratory tract infection. Contact lens wearers should be made aware of the potential risk of CLARE associated with the wearing of contact lenses for extended periods during and subsequent to upper respiratory tract infection.

Acute Disease↗
PubMed1996

The effect of eye closure on protein and complement deposition on Group IV hydrogel contact lenses: relationship to tear flow dynamics.

PURPOSE: This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure. METHODS: Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions. Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90 degrees C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 8.00). Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis. Extracted protein was quantitated and separated by SDS-PAGE. Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted. Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3. Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme. RESULTS: The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C). This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein. In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to lesser extent lysozyme dimer. This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme. C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products. High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye. In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20 +/- .08 versus 0.79 +/- .15 mg/lens (n = 6)). Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2 +/- 0.29 (n = 5) and 0.20 +/- 0.06 microgram/min (n = 4) under open and closed eye conditions respectively. With further wear, deposition eventually reached a steady state. Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot. This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions. CONCLUSIONS: Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompatibility. Findings support the contention that eye closure results in a > 90% reduction in the rate of reflex-type tear secretion.

Adult↗
PubMed1996

Fibronectin in the tear film.

PURPOSE: Fibronectin plays an important role in corneal healing and has been detected previously in the tear film. To investigate the levels of fibronectin in normal human tears, the authors measured and compared fibronectin concentration in open-eye, closed-eye, and reflex tear fluid. The origin of fibronectin in the tear film was investigated by comparing fibronectin concentration in sequentially collected reflex tear samples with the concentrations of total protein and albumin in the same samples. METHODS: Open-eye and closed-eye tears were collected from 11 noncontact lens wearers. From 7 subjects, 20 uninterrupted reflex tear samples (10 microliters each) subsequently were collected, using the sneeze reflex method of stimulation, followed by an additional 10 nonstimulated tear samples (3 microliters each) immediately after cessation of stimulus. Enzyme-linked immunosorbent assays were used to determine fibronectin and albumin concentrations, and bicinchoninic acid protein assays were used to determine total protein concentration in each sample. RESULTS: Fibronectin concentration in open-eye tears (19 +/- 24 eta g/ml, range 3 to 78 eta g/ml) was significantly different (P = 0.004) from that in closed-eyes tears (4127 +/- 3222 eta g/ml, range 1177 to 11384 eta g/ml). In the first 50 microliters of reflex tears, fibronectin concentrations were low (10 +/- 23 eta g/ml), but they increased significantly (P = 0.028) after 100 microliters of reflex tears had been collected (220 +/- 126 eta g/ml). There was a further marked transient increase (767 +/- 946 eta g/ml) after cessation of stimulus. Total protein concentration in the same samples decreased significantly during reflex tear collection compared to open-eye tears, and it increased gradually after cessation of stimulus. Albumin concentration in the same samples, analyzed for two subjects only, showed a pattern similar to that for fibronectin. Dilation of conjunctival blood vessels was noted in all subjects after reflex tear collection. Administration of a topical vasoconstrictor in two subjects eliminated the increase in fibronectin concentration during reflex tearing but did not affect total protein concentration. Under reducing conditions, the molecular mass of fibronectin in open-eye and reflex tears was 240 kDa, identical to commercially available purified plasma fibronectin, whereas fibronectin in closed-eye tears was degraded into small molecular mass fragments. CONCLUSIONS: These findings suggest that fibronectin in tear fluid is derived from plasma and that the increase in concentration in closed-eye and reflex tear fluid is caused by leakage from dilated conjunctival blood vessels.

Adult↗
PubMed1996

Gram-negative bacteria can induce contact lens related acute red eye (CLARE) responses.

PURPOSE: Twelve volunteers participated in a study designed to measure the overnight corneal edema response with a variety of hydrogel contact lenses. During the study four subjects (5 eyes) experienced a contact lens related acute red eye (CLARE) reaction, which manifested as severe ocular pain, photophobia, corneal infiltration, and conjunctival hyperemia. An additional five subjects (7 eyes) developed corneal infiltrates only. Twelve eyes (of 9 subjects) showed no response. METHODS AND RESULTS: Upon microbiological examination of the contact lenses and storage solutions, gram-negative bacteria were isolated in large amounts. The bacteria were identified as Serratia marcescens, Pseudomonas putida, and Pseudomonas aeruginosa. Significantly greater numbers of bacteria were isolated from contact lenses of subjects who experienced CLARE than from the other subjects (P = 0.005) and from the contact lenses of subjects who experienced an adverse reaction (CLARE or infiltrates) than from the other subjects (P < 0.001). The contaminating bacteria are thought to have been introduced to the lens storage vials as a result of lens handling and subsequent failure to disinfect lenses. CONCLUSIONS: This study draws attention to the possible contribution of contaminated lenses and storage cases in contact lens related acute inflammation and specifically implicates gram-negative bacteria, in particular Pseudomonas spp. and Serratia spp., in the inducement of acute inflammatory reactions such as CLARE.

Acute Disease↗