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The application of counter immunoelectrophoresis (CIE) in ocular protein studies
Contact Lens & Anterior Eye 25 (2002) 81–88
The application of counter immunoelectrophoresis (CIE) in ocular protein studies Part II: Kinin activity in the lens wearing eye Aisling M. Mann∗ , Brian J. Tighe Biomaterials Research Unit, School of Engineering and Applied Science, Aston University, Birmingham B4 7ET, UK Received 10 June 2001; received in revised form 20 March 2002; accepted 20 March 2002
Abstract The kinin family are a group of bioactive peptides that are closely involved in the modulation of vascular inflammation and local injury. We have demonstrated here, for the first time, a link between kinin activity and contact lens wear. Protein extracts from daily and extended wear etafilcon A, Group IV, Acuvue lenses (Vistakon), were analysed by counter immunoelectrophoresis. In this way, kinin activity associated with contact lens wear was detected. High molecular weight kininogen was used as the marker protein. In contrast, no kinin activity was detected in the non-lens wearing normal eye. © 2002 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved. Keywords: High molecular weight kininogen; Inflammatory mediators; Contact lens; Tears; Counter immunoelectrophoresis
1. Introduction The kinins are a family of potent bioactive peptides which directly mediate inflammation. This enzyme cascade produces the end product bradykinin, a nonapeptide with many pro-inflammatory functions. The consequences of kinin activation and bradykinin generation includes an increase in vascular permeability, vasodilation, pain, smooth muscle contraction and an ability to stimulate arachidonic acid metabolism [1,2]. Initiation is achieved on the interaction of Hageman factor (Hf) [3], prekallikrein [4,5] and high molecular weight kininogen [6] with a variety of negatively charged species, both natural and synthetic, e.g. Lipid A of endotoxin and glass, respectively which result in Hf (coagulation factor XII) activation and cleavage. The Hf cleavage induces kinin cascade activation through the activation of kallikrein [7], it also activates the intrinsic coagulation system and initiates fibrinolysis. The Hf is the core protein in kinin activation, and central to the continuation of the kinin cascade are the two other plasma proteins, high molecular weight kininogen and prekallikrein. The Hf converts prekallikrein to kallikrein. Kallikrein, an intermediate enzyme, can activate the complement system and acts as a positive feedback activator during the interaction of Hf ∗ Corresponding author. URL: http://www.ceac.aston.ac.uk/biomaterials/biomaterials.html
with the contact surface [8]. It also digests high molecular weight kininogen resulting in the release of the vasoactive peptide bradykinin. A summary of the kinin cascade is shown diagrammatically in Fig. 1. Two forms of plasma kininogen occur, a high molecular weight kininogen (HMWK), at 120 kDa and a low molecular weight kininogen (LMWK) at 68 kDa [9]. HMWK (used here as a marker for kinin activity) is a single chain glycoprotein that, on proteolytic cleavage, gives rise to bradykinin and is bound to the activating moiety, presumably in proximity to the Hf. The concentration of HMWK in plasma has been determined to be in the range of 70–90 g/ml [10,11]. HMWK possesses coagulant activity which depends on the ability of cleaved HMWK to bind anionic surfaces and to associate with prekallikrein [12]. This highlights the importance of the negatively charged contact system activation. Initially, both forms of kininogen were thought to simply be kinin precursors—a stepping stone for the whole cascade, but this would have meant that only 1.6 and 0.8% of the LMWK and HMWK, respectively would have been utilised [13]. In response to this actuality, other functions have been determined—these include cofactor in the coagulation cascade (HMWK) [14,15], an inhibitor of cysteine proteases, such as calpain, and papain (HMWK and LMWK) [16] and a potent anti-adhesive protein which can inhibit the spreading of certain cells on vitronectin or fibrinogen (HMWK) [17]. HMWK also has the ability to
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Fig. 1. Simplification of the kinin pathway.
react with a variety of cell types including platelets, where it acts as a binding site for factor XI [18] and neutrophils, where it acts as an acquired receptor for kallikrein binding which mediates neutrophil degranulation [19]. The kinins have been reported to be involved in oedema formation of hereditary angioedema evidenced by an increase in kallikrein levels found in the serous fluid of blisters induced on the skin [20]. Furthermore, in allergic rhinitis, HMWKs have been detected in nasal secretions [21]. Recent studies have also demonstrated kinin responses in bronchoalveolar fluids in asthmatics [22] and bradykinin has been shown to cause an increase in pulmonary inflation pressure, mucus secretion and breathlessness [23]. The physiological role of the kinin system in these responses remains unclear. It has yet to be seen whether kinin activation (in some or all instances), is a direct cause of symptoms or whether its activation and involvement is as a consequence of an excessive or natural host response. Whatever the cause, the effect of kinins on mucosal surfaces and secretions indicates a possible role of kinins in the ocular environment under certain circumstances. It is well known that deposits on the lens surface can adversely affect successful contact lens wear. These deposits consist primarily of proteins, mucins and lipids from tears, with extraneous substances such as make up and skin lipids also identified. In this study, it is the role of proteins that commands attention because of their capacity for stimulating and mediating immunological reactions, which are usually beneficial to the host. In some circumstances, such a reaction can result in a response that can be detrimental to the host. This paper discusses the interaction of one particular protein, HMWK, with the contact lens. HMWK is regarded here as a marker protein indicating the possible presence of kinin activity. No previous work has been reported on the kinins in tears and lens wear, but because of the increased awareness of plasma protein leakage into tears, the overall change in tear
protein composition and the changes in the physical aspects of the ocular environment experienced during contact lens wear, it was considered logical to investigate its presence.
2. Methods 2.1. Clinical protocol 2.1.1. Daily wear lenses A patient population of 10 was required to wear etafilcon A, Group IV, Acuvue lenses (Vistakon) for 28 days on a daily wear regime using ReNu (Bausch & Lomb) disinfecting cleaner. 2.1.2. Extended wear lenses A different set of patients numbering 22 wore etafilcon A, Group IV, Acuvue lenses (Vistakon) for 7 days and nights only extended wear time period. The use of the Vistakon Acuvue material has the advantage that the lens design, material and fit of the conventional daily wear lens and extended wear lens is almost identical. 2.1.3. Normal basal tear collection Non-stimulated tear samples were collected by microcapillary pipettes from another group of volunteers (n = 10). Tears were taken randomly at different times of the day and over many months, taking in seasonal variation. The tear samples were assayed immediately on collection. All patients were entered into a controlled, randomised and rigidly monitored clinical study. All the lenses were analysed for deposited protein comparing wear time and modality. At the end of each designated wear schedule, all lenses were stored in saline prior to laboratory analysis and refrigerated. No subjects had prior exposure to either the test or control lenses or solutions. Subjects with eye diseases, allergies, insufficient lacrimal secretions, pre-existing ocular
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infections and problems with lens wear were all excluded. The study received prior ethics approval at Aston University and once the requirements of the study had been explained to the subjects, their written, informed consent to take part was obtained. Greater details of the clinical procedures are described in an earlier study [24].
3. Analytical protocol The multifunctional, ubiquitous nature of HMWK, its central role in the kinin system and the availability of commercially produced antibody and antigen (purified protein), determined its use as a marker for kinin related activity. Two main options were available to investigate HMWK using the lens as a source. The choice was to either probe the lens surface or to extract the proteins off the lens. The latter was selected as it enabled us to readily analyse the eluates of the lenses for both HMWK and for other proteins involved in spoilation as part of parallel studies. This procedure required the combined use of an efficient protein extraction technique and immunoassay to investigate the resultant eluate. Counter immunoelectrophoresis (CIE) was the immunoassay of choice which was applied and optimised for the detection and assessment of individual protein species profiles in the extracted eluate [25]. 3.1. Ultra violet (UV) spectrophotometry of lenses for total protein content The initial step in the analysis of the proteins on the contact lens was to measure the total protein levels on the lens prior to analysing the individual proteins deposited. The measurement of total protein by UV was based on an assay originally described in the analysis of deposited proteins on contact lenses [26], measuring protein absorbance at 280 nm. The on-lens UV spectrophotometric assay has proven an invaluable method for calculating the concentration of total protein deposition. This procedure was also fundamental in assessing the extraction efficiency of each lens deposit removal. Individual lens measurements were taken prior to extraction (i), providing total deposition measurements and post extraction (x) in order to calculate the % of protein removed from the lens. i−x × 100 = % of protein extracted i
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efficacy of this method on analysed ex vivo spoilt lenses was 85–90%. It was appreciated that although this method allowed a high extraction efficiency for the etafilcon A lenses, it was not a complete process. However, whilst it was not 100% efficient, for the purpose of this study which was to detect the HMWK presence and not to quantify, it was deemed suitable. Control experiments were performed demonstrating that HMWK was removable by this method. Etafilcon A lenses were in vitro doped with purified HMWK (two-chain) (Sigma) for 1, 7 and 28 days, HMWK was extracted and detected after all three timed intervals. A 1 day in vitro doping was performed to demonstrate the fact that HMWK could be detected by CIE after a short period of exposure of the lens to the protein. 3.3. Tear and lens eluate analysis by counter immunoelectrophoresis (CIE) The CIE assays were used to analyse both the normal basal tear samples and the eluates of the Group IV Acuvue daily and extended wear lenses. A 1.2% agarose and 3% PEG in 1× Tris/Boric acid/EDTA (TBE) buffer was the semi-solid gel matrix of choice. 5 l of the tear sample or eluted lens sample was run against the kinin marker antibody, (goat anti-human HMWK (light chain) (ICN)). The analytes and the antibodies were loaded into a pre-cut Melinex (PSG Group Ltd.) template sheet and allowed to diffuse for 1 h. The gel was then electrophoresed for 1 h on a modified Beckman Paragon electrophoresis system; Barbital buffer (Fluka) was used in the electrophoresis tank. The gels were then washed, dried and stained either with Coomassie blue (ICN) or silver stain (Bio-RAD) ready for analysis and scanning. Although the protein extraction process is a harsh method and consequently, the proteins removed may undergo some degree of denaturation, the use of CIE, which relies on the interaction between the polyclonal antibody and a variety of specific protein epitopes, negates the need for whole unreduced protein. CIE, generally employed in the detection of distinct species in a variety of complex fluids such as serum, has not previously been utilised in lens deposit analysis. This electro-immunodiffusion assay was taken a step further and combined with the above lens extraction method which allowed us to identify the individual nature of each protein involved in lens spoilation and in particular HMWK.
3.2. Protein removal from etafilcon A lenses
4. Results
The optimum lens extraction method was previously found to be a urea/sodium dodecyl sulphate (SDS)/Dl-dithiothretol (DTT)/Tris[hydroxymethyl]aminomethane solution. Each lens to be extracted was heated to 90 ◦ C for 3 h, in 200 l of the extraction solution in a 1 ml eppendorf tube and then allowed to cool gradually. The average extraction
The results of the tear analysis are illustrated in the gel scanned in Fig. 2, which shows the positive lines of precipitation between the tear sample and each respective antibody with the exception of HMWK which was not detected in the normal eye tears within the limits of the assay. This was not totally unexpected as the concentration of HMWK in
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Fig. 2. Normal, non-stimulated open eye tear sample run on counter immunoelectrophoresis.
plasma is quoted at 70–90 g/ml [10,11] and thus, its level in tears would be markedly lower and way below the sensitivity levels of the assay employed. Unfortunately, due to the nature of the clinical study, which was not originally designed with kinin activity investigation in mind, the tear samples tested were not taken from any of the lens wearing subjects under analysis. A collaborative clinical and laboratory study is presently underway which addresses this point, whereby tear samples will be taken pre-lens wear and throughout the patients lens wearing period for a variety of contact lens materials and wear modalities. Preliminary results collected, have shown that subjects presenting with HMWK deposits on the lens had no HMWK in their initial pre-lens wear basal tears. The next step involved the analysis of the etafilcon A lens eluates, using the lens as probe and a source of accumulation.
Extended wear lenses were initially studied because if HMWK was to be detected in conjunction with lens wear, it was hypothesised that extended wear lenses would be a richer source than daily wear lenses. Overnight wear of contact lenses is known to stimulate increased concentrations of serum/plasma influx proteins and of a number of other pro-inflammatory factors, including complement proteins and plasmin [27,28]. 4.1. Extended wear lens analysis The results of one CIE gel performed to investigate HMWK deposition on these lenses is presented in Fig. 3, as an example. From this and similar gels, the rate of occurrence of HMWK deposition in contact lens wearers was calculated; these results presenting the proportion of positive
Fig. 3. CIE of anti-HMWK against six extended wear patient eluates (lenses worn for 7 days and nights).
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Fig. 4. The detection rates of HMWK in the eluates of etafilcon A extended wear lenses (n = 22).
versus negative detection are shown in Fig. 4. 36% of the patients analysed were positive for kinin activity. The results include three patients whose findings were inconclusive where the line of antibody:antigen complex precipitate is blurred. This can occur as a result of residual extraction solution which occasionally appears to precipitate out in the gel, hindering its removal during the washing stage, which can render the immune complex precipitate reaction visually indistinguishable.
4.2. Daily wear lens analysis The results of the daily wear study are summarised in Fig. 5. There was one unresolved reaction which was again due to a masking effect of the residual extraction solution; attempts to dilute this effect did not resolve the result. The percentage of HMWK positive patients was higher than was observed in the extended wear population, that is 60% compared with 36%.
Fig. 5. The detection rates of HMWK in the eluates of etafilcon A daily wear lenses (n = 10).
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5. Discussion HMWK was not found in open eye basal tears, which suggests that it is not present in normal tears of non-lens wearers; it may alternately indicate its presence at levels below the sensitivity of the assay utilised. In marked contrast, the anticipated lens related HMWK influx was clearly observed. HMWK, a glycoprotein previously undetected in normal tears and in lens wear, was discovered in the lens deposits of daily and extended wear etafilcon A lenses. Whether or not HMWK is absent at low levels in normal basal tears, this study specifically shows that the levels of HMWK are enhanced to significant levels under certain lens wear conditions. HMWK was observed in the extracted lens deposits of both conventional daily wear lenses after 28 days wear and 7 day extended wear modalities. As stated earlier it was anticipated that the highest probability of discovering HMWK in lens wear would occur during overnight wear, which has been dubbed a state of sub-clinical inflammation due to the increase in plasma protein content and influx of pro-inflammatory mediators into the tear film [27,28]. Thus, as a follow on step from the extended wear lens analysis, the presence or absence of HMWK in daily wear lenses was determined. This would further implicate the lens (or the lens related response) as a stimulus for kinin activity rather than simply the presence of a lens in the overnight state which alone could be the cause of HMWK presence. The discovery of HMWK accumulation on daily wear lenses without the overnight wear schedule strongly suggested a lens induced kinin influx. The significance of this work is the detection of HMWK and thus kinin activity as a result of lens wear. CIE provided sensitive and reliable results which detected HMWK in the deposits of worn etafilcon A lenses. However, it is interesting to note that not all lens deposits investigated displayed the presence of HMWK. It is important to understand that the distinction between the presence and the absence of HMWK accumulation in the patient population was clear (except in the cases of the small number of unresolved samples), and that it demonstrated definite HMWK positive and HMWK negative population subsets. The difference between the positive and negative detection of HMWK may be due to a number of factors, the greatest of which must be individual patient variation and their response to the lens and applied stress. This itself is interesting since the early presence or absence of HMWK, or indeed any kinin activity, may indicate a patient’s physiological tolerance to specific lens wear. Considering the mode of action of the kinin system in the body and its role in pro-inflammatory functions, their dramatic accumulation in some patients is an indication of stimulation by a certain set of, as yet, unknown conditions. This difference in patient response is a possible warning of existing or potential distress. Further studies are required to obtain a larger sample population to clarify the percentage positive and negative patients under various
conditions. Nevertheless, it is significant that even in this small sample size, a considerable percentage of both sets of patients presented positive for this inflammatory mediator. Regarding the possibility that the detection of kinin activity may be a warning of ocular distress, work done previously has found that bradykinin is released into the tear film of ocular allergic patients; increased provocation with the allergen was associated with an increase in levels of inflammatory mediators including kinins [29]. This highlights the potential of the lens to become directly or indirectly (to be the vector for another foreign body) the causative agent of an adverse response. Additionally, it has been shown that certain corneal epithelial cells have functional bradykinin receptors [30]. This is extremely significant considering that we have shown here that a lens, under certain conditions, is more than capable of providing a means for certain components of the kinin to bind to its surface. The lens provides a means for HMWK to accumulate; HMWK is a precursor to the potent nonapeptide bradykinin, which upon cleavage from HMWK and release from the lens would be in close proximity to the 2 bradykinin receptors on the corneal epithelium which may result in pain or to a lesser extent irritation. The lens induced kinin rich microclimate may at a certain threshold become a clinical concern. In trying to explain the reason(s) for the appearance of HMWK in lens wear, the physical effects of the inserted lens must also be considered. The insertion of a lens into the ocular environment can modify the normal tear dynamics and component distribution; the lens on the cornea can divide the tear film and create a post and pre-lens tear. The surrounding tear film may, for example, exhibit a change in the tear mucin content and distribution and the occurrence of HMWK in lens wear could not be interpreted without acknowledging the aforementioned synergy between the kinins and the mucosal surfaces. There exists a particular affinity and frequent occurrence of the kinins for mucosal sites and their presence in secretory products and thus, there is likely to be a certain association in the kinin family and the mucin content of tears. In contact lens wear, the altered tear layer may thin the post lens mucus layer reducing its lubrication functions between the cornea and the lens, creating friction and thus increasing the irritation caused by the movement of the lens particularly during a blink. Evidence for this lens induced mucin dispersion is seen with the so called ‘mucin ball’ phenomenon which can occur during lens wear [31,32]. This ‘ball’ formation may result in a non-uniform distribution of mucin creating mucin depleted areas which may cause irritation. This irritation may be the stimulus that triggers a host response including a kinin influx and possible activation through the interaction with the negatively charged surface (that is the contact lens). Another interesting point, as stated earlier, is that bradykinin, the end product of the kinin cascade has been shown, in some instances, to stimulate mucus secretion thus easing ocular irritation. In lens wear, it may aid in reducing the aggravation caused by the persistent lens. Additionally, the unwanted excess responses,
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seen at other mucosal sites, may be due to the persistence of the allergen/antigen and in the same way, this may be seen with a contact lens which can be worn continuously for long periods of time. This interest in the association between the kinins and mucin highlights the need to investigate kinin activity with the silicone hydrogels and preliminary work has begun in this area. The consequences of high molecular weight kininogen detection in the ocular environment, and in particular in lens wear, are vast. The detection of new entities, in this case HMWK, provides a greater scope of targets for the development of therapeutics and drugs and pain management in a variety of disorders. Evidence from many in vitro and animal studies have suggested that kinin antagonists are capable of inhibiting kinin related pro-inflammatory effects. The demonstration that kinin antagonists can be used to control the inflammatory response greatly substantiates their involvement in a variety of disorders. In order for the detection of HMWK to be used in the development of therapeutic drugs in the ocular environment it will be necessary to relate HMWK activity to specific aspects of the patient’s response to contact lens wear. Much more work on the kinins in general, and in the ocular environment, is required to provide us with a further insight into the kinin response. We need to understand the effects and consequences of the creation of a microclimate rich in kinin activity on the surface of a contact lens. Consequently, future studies on the involvement of the kinins in lens wear will be performed with the aim of studying a wider patient response to the same lens materials and to study the variation in kinin activation in a range of materials using the same patient base. These, and similar studies, may demonstrate that kinin activation can explain the differences in ocular response to the insertion of a new lens every day, that is daily disposability, versus the reinsertion of a lens with an adapted biofilm. This is potentially an extremely important study in respect of wear modalities. This paper was designed to introduce the detection of kinin activity in the ocular environment and specifically in contact lens wear. It is important to reiterate that the tears provide the first line of defence against potential pathogens and a greater understanding of the host response to the inserted biomaterial will allow us to get closer to our goal to provide a comfortable, safe contact lens. However, as with all new studies, in attempting to provide answers to existing concerns, new questions are posed. It has been suggested here that the presence of HMWK could be an indicator of a negative response, a possible predicative marker of ocular disorder but without clinical data, this rationale could not be backed up. The critical follow up to this study is to work more closely with the clinical observations and to investigate the kinins in contact lens wear over a greater range of materials and patients. One such study underway is the aforementioned work on the silicone hydrogels and other studies are underway to address these and other key issues raised in this paper.
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This study demonstrates a valuable technique for HMWK identification in tear and lens extracts. No HMWK was discovered in the tears of normal open eye non-lens wear subjects. However, when the lens deposits of both of daily and extended wear etafilcon A lenses were analysed, they demonstrated the presence of HMWK in a proportion of extracted lens deposits. This result, overwhelmingly points to the accumulation of HMWK in the ocular environment as a consequence of, or in association with, contact lens wear. References [1] Marceau F, Lussier A, Regoli D, Giroud JP. Pharmacology of kinins: their relevance to tissue injury and inflammation. Gen Pharmacol 1983;14:209–29. [2] Sharma JN, Mohsin SSJ. The role of chemical mediators in the pathogenesis of inflammation with emphasis on the kinin system. Exp Pathol 1990;38:73–96. [3] Margolis J. Activation of plasma by contact with glass: evidence for a common reaction which releases plasma kinin and initiates coagulation. J Physiol 1958;144:1–22. [4] Wuepper KD. Prekallikrein deficiency in man. J Exp Med 1973;138:1345–55. [5] Weiss AS, Gallin JI, Kaplan AP. Fletcher factor deficiency. Abnormalities of coagulation, fibrinolysis, chemotactic activity, and kinin generation attributable to absence of prekallikrein. J Clin Invest 1974;53:622–33. [6] Wuepper KD, Miller DR, LaCombe MJ. Flaujeac trait. Deficiency of human plasma kininogen. J Clin Invest 1975;56:1663–72. [7] Colman RW. Surface-mediated defence reactions: The plasma contact activation system. J Clin Invest 1984;73:1249–53. [8] Cochrane CG, Revak SD, Wuepper KD. Activation of Hageman factor in solid and fluid phase. J Exp Med 1973;138:1564–83. [9] Jacobsen S, Kriz M. Some data on two purified kininogens from human plasma. Br J Pharmacol 1967;29:25–31. [10] Proud D, Pierce JV, Pisano JJ. Radioimmunoassay of human high molecular weight kininogen in normal and deficient plasma. J Clin Invest 1980;92:61–8. [11] Adam A. Human kininogens of low and high molecular mass: quantification by radioimmunoassay and determination of reference values. Clin Chem 1985;31:423–6. [12] Mandle Jr R, Coleman RW, Kaplan AP. Identification of prekallikrein and HMW-kininogen as a circulating complex in human plasma. Proc Natl Acad Sci USA 1976;73:4179–83. [13] DeLaCadena RA, Colman RW. Structure and functions of human kininogens. TIPS 1991;12:272–5. [14] Colman RW, Bagdasarian A, Talamo RC, Scott CF, Seavey M, Guimaraes JA, et al. Williams trait. Human kininogen deficiencies with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J Clin Invest 1975;56:1650–62. [15] Saito H, Ratnoff OD, Waldmann R, Abraham JP. Fitzgerald trait. Deficiency of a hitherto unrecognised agent, Fitzgerald factor, participating in surface mediated reactions of clotting fibrinolysis, generation of kinins. J Clin Invest 1975;55:1082–9. [16] Schmaier AH, Bradford H, Silver LD, Farber A, Scott CF, Schutsky D, et al. High molecular weight kininogen is an inhibitor of platelet calpain. J Clin Invest 1986;77:1565–73. [17] Asakura S, Hurley RW, Skorstengaard K, Ohkubo I, Mosher DF. Inhibition of cell adhesion by high molecular weight kininogen. J Cell Biol 1992;116:465–76. [18] Puri RN, Zhou FX, Hu CJ, Colman RF, Colman RW. High molecular weight kininogen inhibits thrombin induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets. Blood 1991;77:500–7.
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[19] Schapira M, Despland E, Scott CF, Boxer LA, Colman RW. Purified human plasma kallikrein aggregates human blood neutrophils. J Clin Invest 1982;69:1199–202. [20] Proud D, Kaplan AP. Kinin formation: mechanisms and role in inflammatory disorders. Ann Rev Immunol 1988;6:49–83. [21] Proud D, Togias A, Naclerio RM, Crush SA, Norman PS, Lichtenstein LM. Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. J Clin Invest 1983;72: 1678–85. [22] Lukjan H, Hofman J, Kiersnoroska B, Bielawiec M, ChyrekBorowska S. The kinin system in allergic states. Allergy Immunol Band 1972;18:25–30. [23] Farmer SG. Role of kinins in airway diseases. Immunopharmacology 1991;22:1–20. [24] Jones L, Mann A, Evans K, Franklin V, Tighe B. An in vivo comparison of the kinetics of protein and lipid deposition on Group II and Group IV frequent replacement contact lenses. Optom Vis Sci 2000;77:503–10. [25] Mann AM, Tighe BJ. The development of a CIE based assay to investigate the impact of wear regime on the deposition of immunoregulatory proteins. J Am Acad Optom 1999;76: s224.
[26] Karageozian HL, Walden FA, Boghosian MP. Cleaning soft contact lenses. ICLC 1976;3:78–80. [27] Sack RA, Tan KO, Tan A. Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 1992;33:626–40. [28] Tan KO, Sack RA, Holden BA, Swarbrick HA. Temporal sequence changes in the tear film composition during sleep. Curr Eye Res 1993;12:1001–7. [29] Proud D, Sweet J, Stein P, Settipane RA, Kagey-Sobotka A, Friedlaender MH, et al. Inflammatory mediator release on conjunctival provocation of allergic subjects with allergen. J Allergy Clin Immunol 1990;85:896–905. [30] Wiernas TK, Griffin BW, Sharif NA. The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists. Br J Pharmacol 1997;121:649–56. [31] Bourassa S, Benjamin WJ. Transient corneal surface “microdeposits” and associated epithelial surface pits occurring with gel contact lens extended wear. ICLC 1988;15:338–40. [32] Pritchard N, Jones L, Dumbledon K, Fonn D. Epithelial inclusions in association with mucin ball development in high oxygen permeability hydrogel lenses. Optom Vis Sci 2000;77:68–72.
The application of counter immunoelectrophoresis (CIE) in ocular protein studies Part II: Kinin activity in the lens wearing eye Aisling M. Mann∗ , Brian J. Tighe Biomaterials Research Unit, School of Engineering and Applied Science, Aston University, Birmingham B4 7ET, UK Received 10 June 2001; received in revised form 20 March 2002; accepted 20 March 2002
Abstract The kinin family are a group of bioactive peptides that are closely involved in the modulation of vascular inflammation and local injury. We have demonstrated here, for the first time, a link between kinin activity and contact lens wear. Protein extracts from daily and extended wear etafilcon A, Group IV, Acuvue lenses (Vistakon), were analysed by counter immunoelectrophoresis. In this way, kinin activity associated with contact lens wear was detected. High molecular weight kininogen was used as the marker protein. In contrast, no kinin activity was detected in the non-lens wearing normal eye. © 2002 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved. Keywords: High molecular weight kininogen; Inflammatory mediators; Contact lens; Tears; Counter immunoelectrophoresis
1. Introduction The kinins are a family of potent bioactive peptides which directly mediate inflammation. This enzyme cascade produces the end product bradykinin, a nonapeptide with many pro-inflammatory functions. The consequences of kinin activation and bradykinin generation includes an increase in vascular permeability, vasodilation, pain, smooth muscle contraction and an ability to stimulate arachidonic acid metabolism [1,2]. Initiation is achieved on the interaction of Hageman factor (Hf) [3], prekallikrein [4,5] and high molecular weight kininogen [6] with a variety of negatively charged species, both natural and synthetic, e.g. Lipid A of endotoxin and glass, respectively which result in Hf (coagulation factor XII) activation and cleavage. The Hf cleavage induces kinin cascade activation through the activation of kallikrein [7], it also activates the intrinsic coagulation system and initiates fibrinolysis. The Hf is the core protein in kinin activation, and central to the continuation of the kinin cascade are the two other plasma proteins, high molecular weight kininogen and prekallikrein. The Hf converts prekallikrein to kallikrein. Kallikrein, an intermediate enzyme, can activate the complement system and acts as a positive feedback activator during the interaction of Hf ∗ Corresponding author. URL: http://www.ceac.aston.ac.uk/biomaterials/biomaterials.html
with the contact surface [8]. It also digests high molecular weight kininogen resulting in the release of the vasoactive peptide bradykinin. A summary of the kinin cascade is shown diagrammatically in Fig. 1. Two forms of plasma kininogen occur, a high molecular weight kininogen (HMWK), at 120 kDa and a low molecular weight kininogen (LMWK) at 68 kDa [9]. HMWK (used here as a marker for kinin activity) is a single chain glycoprotein that, on proteolytic cleavage, gives rise to bradykinin and is bound to the activating moiety, presumably in proximity to the Hf. The concentration of HMWK in plasma has been determined to be in the range of 70–90 g/ml [10,11]. HMWK possesses coagulant activity which depends on the ability of cleaved HMWK to bind anionic surfaces and to associate with prekallikrein [12]. This highlights the importance of the negatively charged contact system activation. Initially, both forms of kininogen were thought to simply be kinin precursors—a stepping stone for the whole cascade, but this would have meant that only 1.6 and 0.8% of the LMWK and HMWK, respectively would have been utilised [13]. In response to this actuality, other functions have been determined—these include cofactor in the coagulation cascade (HMWK) [14,15], an inhibitor of cysteine proteases, such as calpain, and papain (HMWK and LMWK) [16] and a potent anti-adhesive protein which can inhibit the spreading of certain cells on vitronectin or fibrinogen (HMWK) [17]. HMWK also has the ability to
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Fig. 1. Simplification of the kinin pathway.
react with a variety of cell types including platelets, where it acts as a binding site for factor XI [18] and neutrophils, where it acts as an acquired receptor for kallikrein binding which mediates neutrophil degranulation [19]. The kinins have been reported to be involved in oedema formation of hereditary angioedema evidenced by an increase in kallikrein levels found in the serous fluid of blisters induced on the skin [20]. Furthermore, in allergic rhinitis, HMWKs have been detected in nasal secretions [21]. Recent studies have also demonstrated kinin responses in bronchoalveolar fluids in asthmatics [22] and bradykinin has been shown to cause an increase in pulmonary inflation pressure, mucus secretion and breathlessness [23]. The physiological role of the kinin system in these responses remains unclear. It has yet to be seen whether kinin activation (in some or all instances), is a direct cause of symptoms or whether its activation and involvement is as a consequence of an excessive or natural host response. Whatever the cause, the effect of kinins on mucosal surfaces and secretions indicates a possible role of kinins in the ocular environment under certain circumstances. It is well known that deposits on the lens surface can adversely affect successful contact lens wear. These deposits consist primarily of proteins, mucins and lipids from tears, with extraneous substances such as make up and skin lipids also identified. In this study, it is the role of proteins that commands attention because of their capacity for stimulating and mediating immunological reactions, which are usually beneficial to the host. In some circumstances, such a reaction can result in a response that can be detrimental to the host. This paper discusses the interaction of one particular protein, HMWK, with the contact lens. HMWK is regarded here as a marker protein indicating the possible presence of kinin activity. No previous work has been reported on the kinins in tears and lens wear, but because of the increased awareness of plasma protein leakage into tears, the overall change in tear
protein composition and the changes in the physical aspects of the ocular environment experienced during contact lens wear, it was considered logical to investigate its presence.
2. Methods 2.1. Clinical protocol 2.1.1. Daily wear lenses A patient population of 10 was required to wear etafilcon A, Group IV, Acuvue lenses (Vistakon) for 28 days on a daily wear regime using ReNu (Bausch & Lomb) disinfecting cleaner. 2.1.2. Extended wear lenses A different set of patients numbering 22 wore etafilcon A, Group IV, Acuvue lenses (Vistakon) for 7 days and nights only extended wear time period. The use of the Vistakon Acuvue material has the advantage that the lens design, material and fit of the conventional daily wear lens and extended wear lens is almost identical. 2.1.3. Normal basal tear collection Non-stimulated tear samples were collected by microcapillary pipettes from another group of volunteers (n = 10). Tears were taken randomly at different times of the day and over many months, taking in seasonal variation. The tear samples were assayed immediately on collection. All patients were entered into a controlled, randomised and rigidly monitored clinical study. All the lenses were analysed for deposited protein comparing wear time and modality. At the end of each designated wear schedule, all lenses were stored in saline prior to laboratory analysis and refrigerated. No subjects had prior exposure to either the test or control lenses or solutions. Subjects with eye diseases, allergies, insufficient lacrimal secretions, pre-existing ocular
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infections and problems with lens wear were all excluded. The study received prior ethics approval at Aston University and once the requirements of the study had been explained to the subjects, their written, informed consent to take part was obtained. Greater details of the clinical procedures are described in an earlier study [24].
3. Analytical protocol The multifunctional, ubiquitous nature of HMWK, its central role in the kinin system and the availability of commercially produced antibody and antigen (purified protein), determined its use as a marker for kinin related activity. Two main options were available to investigate HMWK using the lens as a source. The choice was to either probe the lens surface or to extract the proteins off the lens. The latter was selected as it enabled us to readily analyse the eluates of the lenses for both HMWK and for other proteins involved in spoilation as part of parallel studies. This procedure required the combined use of an efficient protein extraction technique and immunoassay to investigate the resultant eluate. Counter immunoelectrophoresis (CIE) was the immunoassay of choice which was applied and optimised for the detection and assessment of individual protein species profiles in the extracted eluate [25]. 3.1. Ultra violet (UV) spectrophotometry of lenses for total protein content The initial step in the analysis of the proteins on the contact lens was to measure the total protein levels on the lens prior to analysing the individual proteins deposited. The measurement of total protein by UV was based on an assay originally described in the analysis of deposited proteins on contact lenses [26], measuring protein absorbance at 280 nm. The on-lens UV spectrophotometric assay has proven an invaluable method for calculating the concentration of total protein deposition. This procedure was also fundamental in assessing the extraction efficiency of each lens deposit removal. Individual lens measurements were taken prior to extraction (i), providing total deposition measurements and post extraction (x) in order to calculate the % of protein removed from the lens. i−x × 100 = % of protein extracted i
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efficacy of this method on analysed ex vivo spoilt lenses was 85–90%. It was appreciated that although this method allowed a high extraction efficiency for the etafilcon A lenses, it was not a complete process. However, whilst it was not 100% efficient, for the purpose of this study which was to detect the HMWK presence and not to quantify, it was deemed suitable. Control experiments were performed demonstrating that HMWK was removable by this method. Etafilcon A lenses were in vitro doped with purified HMWK (two-chain) (Sigma) for 1, 7 and 28 days, HMWK was extracted and detected after all three timed intervals. A 1 day in vitro doping was performed to demonstrate the fact that HMWK could be detected by CIE after a short period of exposure of the lens to the protein. 3.3. Tear and lens eluate analysis by counter immunoelectrophoresis (CIE) The CIE assays were used to analyse both the normal basal tear samples and the eluates of the Group IV Acuvue daily and extended wear lenses. A 1.2% agarose and 3% PEG in 1× Tris/Boric acid/EDTA (TBE) buffer was the semi-solid gel matrix of choice. 5 l of the tear sample or eluted lens sample was run against the kinin marker antibody, (goat anti-human HMWK (light chain) (ICN)). The analytes and the antibodies were loaded into a pre-cut Melinex (PSG Group Ltd.) template sheet and allowed to diffuse for 1 h. The gel was then electrophoresed for 1 h on a modified Beckman Paragon electrophoresis system; Barbital buffer (Fluka) was used in the electrophoresis tank. The gels were then washed, dried and stained either with Coomassie blue (ICN) or silver stain (Bio-RAD) ready for analysis and scanning. Although the protein extraction process is a harsh method and consequently, the proteins removed may undergo some degree of denaturation, the use of CIE, which relies on the interaction between the polyclonal antibody and a variety of specific protein epitopes, negates the need for whole unreduced protein. CIE, generally employed in the detection of distinct species in a variety of complex fluids such as serum, has not previously been utilised in lens deposit analysis. This electro-immunodiffusion assay was taken a step further and combined with the above lens extraction method which allowed us to identify the individual nature of each protein involved in lens spoilation and in particular HMWK.
3.2. Protein removal from etafilcon A lenses
4. Results
The optimum lens extraction method was previously found to be a urea/sodium dodecyl sulphate (SDS)/Dl-dithiothretol (DTT)/Tris[hydroxymethyl]aminomethane solution. Each lens to be extracted was heated to 90 ◦ C for 3 h, in 200 l of the extraction solution in a 1 ml eppendorf tube and then allowed to cool gradually. The average extraction
The results of the tear analysis are illustrated in the gel scanned in Fig. 2, which shows the positive lines of precipitation between the tear sample and each respective antibody with the exception of HMWK which was not detected in the normal eye tears within the limits of the assay. This was not totally unexpected as the concentration of HMWK in
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Fig. 2. Normal, non-stimulated open eye tear sample run on counter immunoelectrophoresis.
plasma is quoted at 70–90 g/ml [10,11] and thus, its level in tears would be markedly lower and way below the sensitivity levels of the assay employed. Unfortunately, due to the nature of the clinical study, which was not originally designed with kinin activity investigation in mind, the tear samples tested were not taken from any of the lens wearing subjects under analysis. A collaborative clinical and laboratory study is presently underway which addresses this point, whereby tear samples will be taken pre-lens wear and throughout the patients lens wearing period for a variety of contact lens materials and wear modalities. Preliminary results collected, have shown that subjects presenting with HMWK deposits on the lens had no HMWK in their initial pre-lens wear basal tears. The next step involved the analysis of the etafilcon A lens eluates, using the lens as probe and a source of accumulation.
Extended wear lenses were initially studied because if HMWK was to be detected in conjunction with lens wear, it was hypothesised that extended wear lenses would be a richer source than daily wear lenses. Overnight wear of contact lenses is known to stimulate increased concentrations of serum/plasma influx proteins and of a number of other pro-inflammatory factors, including complement proteins and plasmin [27,28]. 4.1. Extended wear lens analysis The results of one CIE gel performed to investigate HMWK deposition on these lenses is presented in Fig. 3, as an example. From this and similar gels, the rate of occurrence of HMWK deposition in contact lens wearers was calculated; these results presenting the proportion of positive
Fig. 3. CIE of anti-HMWK against six extended wear patient eluates (lenses worn for 7 days and nights).
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Fig. 4. The detection rates of HMWK in the eluates of etafilcon A extended wear lenses (n = 22).
versus negative detection are shown in Fig. 4. 36% of the patients analysed were positive for kinin activity. The results include three patients whose findings were inconclusive where the line of antibody:antigen complex precipitate is blurred. This can occur as a result of residual extraction solution which occasionally appears to precipitate out in the gel, hindering its removal during the washing stage, which can render the immune complex precipitate reaction visually indistinguishable.
4.2. Daily wear lens analysis The results of the daily wear study are summarised in Fig. 5. There was one unresolved reaction which was again due to a masking effect of the residual extraction solution; attempts to dilute this effect did not resolve the result. The percentage of HMWK positive patients was higher than was observed in the extended wear population, that is 60% compared with 36%.
Fig. 5. The detection rates of HMWK in the eluates of etafilcon A daily wear lenses (n = 10).
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5. Discussion HMWK was not found in open eye basal tears, which suggests that it is not present in normal tears of non-lens wearers; it may alternately indicate its presence at levels below the sensitivity of the assay utilised. In marked contrast, the anticipated lens related HMWK influx was clearly observed. HMWK, a glycoprotein previously undetected in normal tears and in lens wear, was discovered in the lens deposits of daily and extended wear etafilcon A lenses. Whether or not HMWK is absent at low levels in normal basal tears, this study specifically shows that the levels of HMWK are enhanced to significant levels under certain lens wear conditions. HMWK was observed in the extracted lens deposits of both conventional daily wear lenses after 28 days wear and 7 day extended wear modalities. As stated earlier it was anticipated that the highest probability of discovering HMWK in lens wear would occur during overnight wear, which has been dubbed a state of sub-clinical inflammation due to the increase in plasma protein content and influx of pro-inflammatory mediators into the tear film [27,28]. Thus, as a follow on step from the extended wear lens analysis, the presence or absence of HMWK in daily wear lenses was determined. This would further implicate the lens (or the lens related response) as a stimulus for kinin activity rather than simply the presence of a lens in the overnight state which alone could be the cause of HMWK presence. The discovery of HMWK accumulation on daily wear lenses without the overnight wear schedule strongly suggested a lens induced kinin influx. The significance of this work is the detection of HMWK and thus kinin activity as a result of lens wear. CIE provided sensitive and reliable results which detected HMWK in the deposits of worn etafilcon A lenses. However, it is interesting to note that not all lens deposits investigated displayed the presence of HMWK. It is important to understand that the distinction between the presence and the absence of HMWK accumulation in the patient population was clear (except in the cases of the small number of unresolved samples), and that it demonstrated definite HMWK positive and HMWK negative population subsets. The difference between the positive and negative detection of HMWK may be due to a number of factors, the greatest of which must be individual patient variation and their response to the lens and applied stress. This itself is interesting since the early presence or absence of HMWK, or indeed any kinin activity, may indicate a patient’s physiological tolerance to specific lens wear. Considering the mode of action of the kinin system in the body and its role in pro-inflammatory functions, their dramatic accumulation in some patients is an indication of stimulation by a certain set of, as yet, unknown conditions. This difference in patient response is a possible warning of existing or potential distress. Further studies are required to obtain a larger sample population to clarify the percentage positive and negative patients under various
conditions. Nevertheless, it is significant that even in this small sample size, a considerable percentage of both sets of patients presented positive for this inflammatory mediator. Regarding the possibility that the detection of kinin activity may be a warning of ocular distress, work done previously has found that bradykinin is released into the tear film of ocular allergic patients; increased provocation with the allergen was associated with an increase in levels of inflammatory mediators including kinins [29]. This highlights the potential of the lens to become directly or indirectly (to be the vector for another foreign body) the causative agent of an adverse response. Additionally, it has been shown that certain corneal epithelial cells have functional bradykinin receptors [30]. This is extremely significant considering that we have shown here that a lens, under certain conditions, is more than capable of providing a means for certain components of the kinin to bind to its surface. The lens provides a means for HMWK to accumulate; HMWK is a precursor to the potent nonapeptide bradykinin, which upon cleavage from HMWK and release from the lens would be in close proximity to the 2 bradykinin receptors on the corneal epithelium which may result in pain or to a lesser extent irritation. The lens induced kinin rich microclimate may at a certain threshold become a clinical concern. In trying to explain the reason(s) for the appearance of HMWK in lens wear, the physical effects of the inserted lens must also be considered. The insertion of a lens into the ocular environment can modify the normal tear dynamics and component distribution; the lens on the cornea can divide the tear film and create a post and pre-lens tear. The surrounding tear film may, for example, exhibit a change in the tear mucin content and distribution and the occurrence of HMWK in lens wear could not be interpreted without acknowledging the aforementioned synergy between the kinins and the mucosal surfaces. There exists a particular affinity and frequent occurrence of the kinins for mucosal sites and their presence in secretory products and thus, there is likely to be a certain association in the kinin family and the mucin content of tears. In contact lens wear, the altered tear layer may thin the post lens mucus layer reducing its lubrication functions between the cornea and the lens, creating friction and thus increasing the irritation caused by the movement of the lens particularly during a blink. Evidence for this lens induced mucin dispersion is seen with the so called ‘mucin ball’ phenomenon which can occur during lens wear [31,32]. This ‘ball’ formation may result in a non-uniform distribution of mucin creating mucin depleted areas which may cause irritation. This irritation may be the stimulus that triggers a host response including a kinin influx and possible activation through the interaction with the negatively charged surface (that is the contact lens). Another interesting point, as stated earlier, is that bradykinin, the end product of the kinin cascade has been shown, in some instances, to stimulate mucus secretion thus easing ocular irritation. In lens wear, it may aid in reducing the aggravation caused by the persistent lens. Additionally, the unwanted excess responses,
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seen at other mucosal sites, may be due to the persistence of the allergen/antigen and in the same way, this may be seen with a contact lens which can be worn continuously for long periods of time. This interest in the association between the kinins and mucin highlights the need to investigate kinin activity with the silicone hydrogels and preliminary work has begun in this area. The consequences of high molecular weight kininogen detection in the ocular environment, and in particular in lens wear, are vast. The detection of new entities, in this case HMWK, provides a greater scope of targets for the development of therapeutics and drugs and pain management in a variety of disorders. Evidence from many in vitro and animal studies have suggested that kinin antagonists are capable of inhibiting kinin related pro-inflammatory effects. The demonstration that kinin antagonists can be used to control the inflammatory response greatly substantiates their involvement in a variety of disorders. In order for the detection of HMWK to be used in the development of therapeutic drugs in the ocular environment it will be necessary to relate HMWK activity to specific aspects of the patient’s response to contact lens wear. Much more work on the kinins in general, and in the ocular environment, is required to provide us with a further insight into the kinin response. We need to understand the effects and consequences of the creation of a microclimate rich in kinin activity on the surface of a contact lens. Consequently, future studies on the involvement of the kinins in lens wear will be performed with the aim of studying a wider patient response to the same lens materials and to study the variation in kinin activation in a range of materials using the same patient base. These, and similar studies, may demonstrate that kinin activation can explain the differences in ocular response to the insertion of a new lens every day, that is daily disposability, versus the reinsertion of a lens with an adapted biofilm. This is potentially an extremely important study in respect of wear modalities. This paper was designed to introduce the detection of kinin activity in the ocular environment and specifically in contact lens wear. It is important to reiterate that the tears provide the first line of defence against potential pathogens and a greater understanding of the host response to the inserted biomaterial will allow us to get closer to our goal to provide a comfortable, safe contact lens. However, as with all new studies, in attempting to provide answers to existing concerns, new questions are posed. It has been suggested here that the presence of HMWK could be an indicator of a negative response, a possible predicative marker of ocular disorder but without clinical data, this rationale could not be backed up. The critical follow up to this study is to work more closely with the clinical observations and to investigate the kinins in contact lens wear over a greater range of materials and patients. One such study underway is the aforementioned work on the silicone hydrogels and other studies are underway to address these and other key issues raised in this paper.
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This study demonstrates a valuable technique for HMWK identification in tear and lens extracts. No HMWK was discovered in the tears of normal open eye non-lens wear subjects. However, when the lens deposits of both of daily and extended wear etafilcon A lenses were analysed, they demonstrated the presence of HMWK in a proportion of extracted lens deposits. This result, overwhelmingly points to the accumulation of HMWK in the ocular environment as a consequence of, or in association with, contact lens wear. References [1] Marceau F, Lussier A, Regoli D, Giroud JP. Pharmacology of kinins: their relevance to tissue injury and inflammation. Gen Pharmacol 1983;14:209–29. [2] Sharma JN, Mohsin SSJ. The role of chemical mediators in the pathogenesis of inflammation with emphasis on the kinin system. Exp Pathol 1990;38:73–96. [3] Margolis J. Activation of plasma by contact with glass: evidence for a common reaction which releases plasma kinin and initiates coagulation. J Physiol 1958;144:1–22. [4] Wuepper KD. Prekallikrein deficiency in man. J Exp Med 1973;138:1345–55. [5] Weiss AS, Gallin JI, Kaplan AP. Fletcher factor deficiency. Abnormalities of coagulation, fibrinolysis, chemotactic activity, and kinin generation attributable to absence of prekallikrein. J Clin Invest 1974;53:622–33. [6] Wuepper KD, Miller DR, LaCombe MJ. Flaujeac trait. Deficiency of human plasma kininogen. J Clin Invest 1975;56:1663–72. [7] Colman RW. Surface-mediated defence reactions: The plasma contact activation system. J Clin Invest 1984;73:1249–53. [8] Cochrane CG, Revak SD, Wuepper KD. Activation of Hageman factor in solid and fluid phase. J Exp Med 1973;138:1564–83. [9] Jacobsen S, Kriz M. Some data on two purified kininogens from human plasma. Br J Pharmacol 1967;29:25–31. [10] Proud D, Pierce JV, Pisano JJ. Radioimmunoassay of human high molecular weight kininogen in normal and deficient plasma. J Clin Invest 1980;92:61–8. [11] Adam A. Human kininogens of low and high molecular mass: quantification by radioimmunoassay and determination of reference values. Clin Chem 1985;31:423–6. [12] Mandle Jr R, Coleman RW, Kaplan AP. Identification of prekallikrein and HMW-kininogen as a circulating complex in human plasma. Proc Natl Acad Sci USA 1976;73:4179–83. [13] DeLaCadena RA, Colman RW. Structure and functions of human kininogens. TIPS 1991;12:272–5. [14] Colman RW, Bagdasarian A, Talamo RC, Scott CF, Seavey M, Guimaraes JA, et al. Williams trait. Human kininogen deficiencies with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor-dependent pathways. J Clin Invest 1975;56:1650–62. [15] Saito H, Ratnoff OD, Waldmann R, Abraham JP. Fitzgerald trait. Deficiency of a hitherto unrecognised agent, Fitzgerald factor, participating in surface mediated reactions of clotting fibrinolysis, generation of kinins. J Clin Invest 1975;55:1082–9. [16] Schmaier AH, Bradford H, Silver LD, Farber A, Scott CF, Schutsky D, et al. High molecular weight kininogen is an inhibitor of platelet calpain. J Clin Invest 1986;77:1565–73. [17] Asakura S, Hurley RW, Skorstengaard K, Ohkubo I, Mosher DF. Inhibition of cell adhesion by high molecular weight kininogen. J Cell Biol 1992;116:465–76. [18] Puri RN, Zhou FX, Hu CJ, Colman RF, Colman RW. High molecular weight kininogen inhibits thrombin induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets. Blood 1991;77:500–7.
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[19] Schapira M, Despland E, Scott CF, Boxer LA, Colman RW. Purified human plasma kallikrein aggregates human blood neutrophils. J Clin Invest 1982;69:1199–202. [20] Proud D, Kaplan AP. Kinin formation: mechanisms and role in inflammatory disorders. Ann Rev Immunol 1988;6:49–83. [21] Proud D, Togias A, Naclerio RM, Crush SA, Norman PS, Lichtenstein LM. Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. J Clin Invest 1983;72: 1678–85. [22] Lukjan H, Hofman J, Kiersnoroska B, Bielawiec M, ChyrekBorowska S. The kinin system in allergic states. Allergy Immunol Band 1972;18:25–30. [23] Farmer SG. Role of kinins in airway diseases. Immunopharmacology 1991;22:1–20. [24] Jones L, Mann A, Evans K, Franklin V, Tighe B. An in vivo comparison of the kinetics of protein and lipid deposition on Group II and Group IV frequent replacement contact lenses. Optom Vis Sci 2000;77:503–10. [25] Mann AM, Tighe BJ. The development of a CIE based assay to investigate the impact of wear regime on the deposition of immunoregulatory proteins. J Am Acad Optom 1999;76: s224.
[26] Karageozian HL, Walden FA, Boghosian MP. Cleaning soft contact lenses. ICLC 1976;3:78–80. [27] Sack RA, Tan KO, Tan A. Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 1992;33:626–40. [28] Tan KO, Sack RA, Holden BA, Swarbrick HA. Temporal sequence changes in the tear film composition during sleep. Curr Eye Res 1993;12:1001–7. [29] Proud D, Sweet J, Stein P, Settipane RA, Kagey-Sobotka A, Friedlaender MH, et al. Inflammatory mediator release on conjunctival provocation of allergic subjects with allergen. J Allergy Clin Immunol 1990;85:896–905. [30] Wiernas TK, Griffin BW, Sharif NA. The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists. Br J Pharmacol 1997;121:649–56. [31] Bourassa S, Benjamin WJ. Transient corneal surface “microdeposits” and associated epithelial surface pits occurring with gel contact lens extended wear. ICLC 1988;15:338–40. [32] Pritchard N, Jones L, Dumbledon K, Fonn D. Epithelial inclusions in association with mucin ball development in high oxygen permeability hydrogel lenses. Optom Vis Sci 2000;77:68–72.