Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

generation topical ocular antihistamines, as inhibitors of histamine-stimulated cytokine synthesis by conjunctival epithelial cells (Yanni et al., 1999a).

Levocabastine is a cyclohexylpiperidine derivative that has a slow rate of dissociation from histamine H1 receptors. A dissociation half-life of 116 minutes has been published (Dechant and Goa, 1991). This characteristic provides enhanced duration of action compared with the first generation antihistamines discussed above. Ocular allergen challenge studies confirm the longer duration of action, which is reported to be at least 4 hours in most studies (Noble and McTavish, 1995). While levocabastine interacts with the histamine H1 receptor, Ki values have also been published for interactions with dopamine D2, α1 adrenergic, and serotonin 5HT2 receptors (Dechant and Goa, 1991). Although these Ki values are higher than the value for histamine receptor binding, they are below 10 μM. This becomes relevant with topical ocular dosing because drug preparations typically contain 250–1000 μg/mL (0.025–0.1%) of active ingredient, which translates into near mM exposures on the ocular surface. Emedastine, a benzimidazole, is a potent, selective histamine H1 antagonist. In vitro receptor binding and second messenger studies have shown that the drug has a dissociation constant of 1.3nM for the H1 receptor and considerably weaker affinity for the H2 and H3 receptors. Comparative data for levocabastine, pheniramine, and antazoline demonstrate emedastine’s greater selectivity for the H1 receptor (Sharif et al., 1994a). Additionally, emedastine did not interact with 38 non-histamine receptors (Sharif et al., 1994b), and provided a greater degree of specificity for the histamine receptor, as well as enhanced selectivity compared with other anti-histamines. The in vitro potency reported was translated into significant in vivo efficacy following topical ocular administration. Emedastine was shown to be significantly more potent than pyrilamine, levocabastine, pheniramine, and

antazoline in models of histamine and allergen induced conjunctivitis. The duration of activity noted in these preclinical studies was 4–8 hours (Yanni et al., 1994), similar to that of levocabastine reported previously.

It has been established that histamine H1 antagonists provide effective therapy for the pruritis associated with allergic responses. However, mast cell activation results in the release/production of a variety of proinflammatory mediators along with histamine. These include eicosanoids, cytokines, and chemotactic molecules that participate in the generation of the signs and symptoms of allergic conjunctivitis. Therefore, to provide maximum therapy, a drug has to antagonize a number of diverse ligand/receptor interactions, which is a difficult medicinal chemistry task. Alternatively, a drug may displace histamine from its H1 receptor to provide acute symptomatic relief, while preventing subsequent mast cell activation and release of mediators. Drugs with both of these activities have become available, and are currently the most commonly prescribed medications for seasonal and perennial allergic conjunctivitis. Olopatadine was the first compound in the dual action class to be approved for the treatment of allergic conjunctivitis. Subsequently, ketotifen, azelastine, and epinastine have also been approved in this class.

Olopatadine is a novel dibenzoxepine that antagonizes histamine H1 receptor activation and prevents immunologically stimulated pro-inflammatory mediator release from human conjunctival mast cells (Yanni et al., 1996; Sharif et al., 1996c). The drug’s pharmacology, pharmacokinetic and clinical properties have been reviewed by Ohmori et al. in 2002 and 2004. Of particular note is the drug’s ability to inhibit the release of vaso-active mediators, eicosanoids, and cytokines from human conjunctival mast cells (Sharif et al., 1996b; Cook et al., 2000) without perturbing cell membrane integrity. Work reported by Brockman et al. (2003) evaluated the effect of olopatadine and other compounds with

dual activity on membrane interactions using model and natural cell membranes. The authors noted that olopatadine was unique among the antihistaminic agents tested because it demonstrated low intrinsic surface activity, thus limiting its interaction with membranes and subsequently not damaging membrane integrity.

Olopatadine’s ability to prevent mast cell degranulation has been compared to the effect of the first generation cromolyn-like mast cell stabilizers using human conjuctival mast cells. Results from these experiments demonstrated olopatadine’s superior mast cell stabilizing effect in comparison with cromolyn, nedocromil, and pemirolast (Yanni et al., 1997). The ability to prevent release of mast cell mediators and limit exposure of surrounding cells to these substances has important implications for the treatment of allergic conjunctivitis. Human conjunctival epithelial cells express functional histamine H1 receptors (Sharif et al., 1996a). Exposure of these cells to histamine results in the secretion of IL-6, IL-8, and GM-CSF (Weimer et al., 1998). Olopatadine significantly inhibits histamine stimulated epithelial cell cytokine production at concentrations an order of magnitude lower than the intrinsic anti-histaminic activity of the drug, suggesting an independent effect upon cytokine synthesis (Yanni et al., 1999b). In an elegant set of experiments, Cook et al. (2001a) demonstrated that human conjunctival mast cells release TNFα upon immunological challenge. This cytokine upregulates human conjunctival epithelial cell ICAM-1 expression. Pre-incubation of mast cells with olopatadine significantly blocked upregulation of ICAM-1 expression on conjunctival epithelial cells induced by mast cell culture supernatants, indicating that preventing mast cell mediator release reduces effects on epithelial cells. Importantly, ICAM-1 expression was restored by adding exogenous TNFα to the olopatadine treated mast cell supernatants, indicating olopatadine’s effect resulted from inhibition of mediator release, and not antagonism of TNFα.

Ketotifen was first described as an antiasthmatic drug. It has been reported to inhibit mast cell mediator release (Schoch, 2003) and possess potent anti-histaminic activity. The drug has also been reported to decrease the accumulation of eosinophils in the airway after respiratory challenge. A review of the pharmacodynamic and pharmacokinetic properties of ketotifen has been published (Grant et al., 1990). Azelastine is another drug in the dual mechanism class that was initially developed to treat respiratory allergy. This drug is a potent histamine H1 receptor antagonist with activity in both in vitro and in vivo studies. In vitro studies in rat and guinea pig mast cells have shown azelastine was capable of inhibiting mediator release from these cell types (McTavish and Sorkin, 1989). The drug’s activity on mature, terminally differentiated human mast cell populations relevant to seasonal allergic conjunctivitis is not available in the peer reviewed literature (Cook et al., 2002).

Epinastine is the most recent drug approved in this category. It, like the other members of the group, is a potent histamine H1 receptor antagonist. Matsukura et al. (2003) reported epinastine inhibited eosinophil chemotaxis in atopic dermatitis, suggesting an anti-inflammatory effect. A review of epinastine’s pharmacology is available (Tasaka, 2000).

The clinical efficacy of this class of agents has been widely reported. Rossenwasser et al. (2005) reviewed available clinical literature referenced in MEDLINE. They noted that the compounds were effective and that olopatadine appeared to be clinically superior. This is consistent with olopatadine’s broader approved indication for all signs and symptoms of SAC and PAC, compared to indications for treating pruritis for other members of this class.

F. Drug Discovery

The information provided above suggests two primary paths are available for

drug discovery research to improve upon these existing therapies. These paths are improved mast cell stabilization effects and improved anti-inflammatory effects. Data suggests that olopatadine is a more effective mast cell stabilizer than other members of this class, and this has been touted as the reason for a broader indication for this drug. This provides direct impetus to identify and develop more effective compounds capable of preventing and attenuating mast cell activation. In a recent paper, Cook et al. (2006) investigated whether tears collected from allergic subjects could promote eosinophil binding to human conjunctival epithelial cells. Tears were collected from olopatadine treated eyes and nontreated eyes. These investigators reported that tears collected from allergic subjects contained bioactivity capable of upregulating eosinophil adhesion to epithelial cells, and that tears from olopatadine treated eyes were significantly less (43%) able to promote eosinophil adhesion. These data suggest mast cell activation results in the secretion into tears of substances capable of promoting an inflammatory effect that can be decreased by treatment with an effective mast cell stabilizer. This provides clinical evidence that preventing mast cell activation will provide broader therapeutic effects than simply preventing ocular itching.

A molecular target that holds promise because it appears to be critical for mast cell degranulation induced by antigen binding to the IgE receptor is spleen tyrosine kinase, referred to as Syk. The Syk kinase was first described in 1991 by Taniguchi et al. Within 2 years this 72 kDa tyrosine kinase was demonstrated to be associated with the IgE receptor on rat tumor mast cells (Hutchcroft et al., 1992, Benhamou et al., 1993). Syk plays a pivotal role in high affinity IgE receptor signaling in mast cells. Tyrosine phosphorylation of multiple cellular substrates occurs within 5–15 seconds of contact with a multivalent antigen. The most prominent kinase

involved in these phosphorylation reactions is Syk (Kinet et al., 1996). Activated Syk in turn phosphorylates other enzymes, including phospholipase CγPLCγ), phos- phatidylinositol-3 kinase, protein kinase C, and others. Activated PLCγ catalyzes cleavage of PIP2 into DAG and IP3. IP3 binds to the IP3 receptor Ca channel in the endoplasmic reticulum membrane and causes an increase in free Ca levels. The free Ca , along with DAG, activates PKCs that activate the JNK pathway, resulting in cytokine production (Luskova and Draber, 2004). The major steps in the signaling pathway are presented in Figure 11.1. The importance of Syk in mast cell activation has been emphasized by the studies of Costello et al. (1996) and Zhang et al. (1996). Syk deficient fetal liver derived mast cells failed to degranulate, synthesize leukotrienes, and secrete cytokines when stimulated through the Fcε receptor. A Syk deficient rat basophilic leukemia RBL-2H3 cell variant was identified that did not release histamine upon IgE receptor aggregation. These cloned cells were then reconstituted by transfection to express stable Syk. In these cells, Fcε receptor aggregation resulted in phosphorylation of phospholipase C, and increase in intracellular free Ca and histamine release.

Currently, a number of pharmaceutical companies are attempting to develop Syk inhibitors for use in allergic diseases. Bayer’s BAY-61-3006 (Figure 11.2) inhibits Syk kinase activity at nM concentrations. When administered orally to animals used in models of immediate hypersensitivity, the drug blocked passive cutaneous anaphylaxis at a dose of 3 mg/kg (Yamamoto et al., 2003). Rigel and Pfizer have recently announced a collaboration to develop Syk kinase inhibitors for allergic airway disease. News reports have also noted that investigators are evaluating the possibility of using small inhibitory RNAs (siRNA) targeted at Syk kinase for respiratory allergic diseases. Whether any of the antiSyk compounds will be developed into

an effective anti-allergy therapy useful for topical ocular administration remains to be determined. However, inhibition of antigen-induced mast cell activation at nM concentrations suggests promise resides in this approach.

The second pathway mentioned above to improve therapies for SAC and PAC is the enhancement of anti-inflammatory efficacy. Corticosteroids have been used to treat SAC and PAC and have dramatic

anti-inflammatory effects. Unfortunately, they also have serious ocular side effects, including intraocular pressure increases and cataractogenesis. Leonardi (2005) wrote, “Corticosteroids should be the last choice in treating allergic disease. They should be avoided in seasonal and perennial allergic conjunctivitis, although corticosteroids present an attractive target for research and therapy.” The currently approved drugs provide a level of

anti-inflammatory activity as noted by the reports of their effects upon cytokine synthesis, as well as leukocyte migration and accumulation noted above (Yanni et al., 1999b; Schoch, 2003; Matsukura et al., 2003).

An approach that is relevant to treating Type 1 immediate hypersensitivity reactions targets histamine binding to the histamine H4 receptor. Oda et al. (2000) cloned and characterized this novel histamine receptor and found that it was preferentially expressed in leukocytes. Hofstra et al. (2003) demonstrated that histamine induced chemotaxis in mouse mast cells without affecting degranulation. They also demonstrated that mast cells harvested from H4 receptor deficient mice did not migrate toward histamine, providing direct proof in mice that histamine acting at the H4 receptor can stimulate migration of mast cells. This allows us to speculate that histamine binding to this receptor on mast cells can lead to the accumulation of mast cells in tissues where histamine is being released, resulting in an amplification of the allergic response. Daugherty (2004) suggested that histamine binding to the H4 receptor on eosinophils could prime these cells to other chemo-attractants, making them hyper-responsive to concentrations encountered in the local tissue environment. He presented reports demonstrating that pre-incubation of eosinophils with histamine resulted in enhanced migration of these cells toward eotaxin and eotaxin-2. More recently, histamine H4 receptor stimulation has been reported to induce human monocyte derived dendritic cell chemotaxis (Gutzmer et al., 2005). Dunford et al. (2006) studied the effect of histamine in H4 deficient mice and mice treated with H4 antagonists. Mice lacking the receptor exhibited decreased allergic lung inflammation with fewer numbers of infiltrating eosinophils and lymphocytes, and a decreased Th2 response. Ex vivo stimulation of T-cells showed decreases in a number of interleukin levels. In vitro studies indicated

that blockade of the H4 receptor on dendritic cells limited their ability to induce Th2 responses in T-cells, suggesting modulating effects upon T-cell activation. The reports noted above suggest that therapies capable of antagonizing agonist binding at the novel histamine H4 receptor may provide significant anti-inflammatory benefits.

In order to fully deploy drug discovery technology aimed at this target, the existence of non-human species homologs of the receptor had to be identified and characterized. Liu et al. (2001) cloned the rat, mouse, and guinea pig cDNAs corresponding to the human histamine H4 receptor. Their results suggest that the identification of potential new drugs capable of antagonizing this receptor will require significant experimentation. The authors found that the H4 sequences for rat, mouse, and guinea pig were significantly different from human H4 sequences. Only 65–69% homology was discovered. Human and guinea pig receptors displayed the highest binding affinity for histamine (KD 5 nM), while rat and mouse were 136 nM and 42 nM, respectively. Differences also exist in binding affinities for known H3/H4 ligands among the species H4 receptor homologs, and differences were described in the signal transduction responses of the homologs.

Toward this end, Thurmond et al. (2004) reported the production and biological results from the pharmacological evaluation of the first histamine H4 antagonist, JNJ 7777120 (Figure 11.3). This compound has a reported Ki of 4.5 nM vs the human H4 receptor and is 1000-fold selective over the other histamine receptors. The compound

N N

N

CH3

JNJ7777120

FIGURE 11.3 H4 antagonist

was equipotent against human, rat, and mouse receptors. The authors reported that JNJ7777120 blocked histamine induced chemotaxis and calcium influx in mouse bone marrow derived mast cells. A sample of this compound was synthesized and evaluated in a model of both acute and late phase allergic conjunctivitis using topical ocular dosing in the laboratory of one of the authors (JMY). Results indicated that eosinophil influx into tissue 24 hours after acute allergen challenge was significantly reduced when the H4 antagonist was applied prior to antigen challenge. However, no effect was noted on the acute symptoms of the allergic response. It is the acute symptoms of SAC and PAC that motivate patients to visit their physician. If the H4 antagonists do not attenuate the acute effects of the allergic response, their use as standalone therapies for these indications may be limited. Their possible use in combination with drugs known to inhibit the acute allergic response, or the synthesis of compounds capable of antagonizing both histamine H1 and H4 induced biological effects, remains a possibility. Synthesis and characterization of other novel antagonists will be required before the class’s utility is understood.

III. CONJUNCTIVITIS

Three other atopic ocular diseases are grouped with SAC and PAC in ocular allergy texts. They are: giant papillary conjunctivitis (GPC); atopic keratoconjunctivitis (AKC); and vernal keratoconjunctivitis (VKC). The clinical descriptions of these conditions, associated immunopathology and pathophysiology are described in the following sections.

A. Giant Papillary Conjunctivitis

Giant papillary conjunctivitis (GPC) is a chronic inflammatory process leading to the production of giant papillae on the tarsal conjunctiva lining of the upper eyelids.

Most often associated with soft contact lens wear, GPC has been reported in patients wearing soft, hard, and rigid gas-permeable contact lenses, as well as in patients with ocular prostheses and exposed sutures in contact with the conjunctiva. GPC may affect as many as 20% of soft contact lens wearers (Allansmith et al., 1977). Those people wearing regular (as opposed to disposable) soft contact lenses are at least 10 times more susceptible to GPC than rigid (gas-permeable) contact lens wearers. Those patients wearing daily-wear disposable contact lenses and those wearing rigid contact lenses are about equally affected. Patients who wear disposable contact lenses during sleep are probably 3 times more likely to have GPC symptoms than if the lenses are removed daily. Patients with asthma, hay fever, or animal allergies may be at greater risk for GPC (Begley et al., 1990).

1. Clinical parameters

Symptoms of GPC include ocular itching after lens removal, redness, burning, increased mucus discharge in the morning, photophobia, and decreased contact lens tolerance. Blurred vision can result from deposits on the contact lens, or from displacement of the contact lens secondary to the superior eyelid papillary hypertrophy. Initial presentation may occur months or even years after the patient has begun wearing contact lenses.

2. Pathophysiology

The onset of GPC may be the result of mechanical trauma secondary to contact lens fit or a lens edge causing chronic irritation of the upper eyelid with each blink. It is more likely, however, that a build-up of “protein” on the surface of the contact lens causes an allergic reaction in the eyelid tissue (Allansmith, 1987; Tan et al., 2002). Tear clearance from the ocular surface of GPC patients is decreased compared to normals, and this may allow the protein in the tear film longer contact time with the

contact lens (Chang and Chang, 2001). As with AKC and VKC, tissue biopsies are the primary source of data on the pathophysiology of this GPC. Many of the published studies concerning mast cell involvement in GPC contrast the disease with VKC. Like VKC, conjunctival biopsies in GPC are found to have mast cells of the MCT type in the conjunctival epithelium. However, there is no significant increase in mast cells in the substantia propria, and therefore no overall increases in number of mast cells present in the conjunctival tissue (Irani et al., 1986). Interestingly, while increased histamine is measured in tears in patients with VKC, patients with GPC have normal tear histamine levels (Abelson et al., 1980; Allansmith et al., 1977). This can be partially explained from electron microscopy data on biopsies from patients with GPC that has revealed less mast cell degranulation (30%) than is observed in patients with VKC (80%) (Henriquez et al., 1981). Tryptase has also been found in the tears from patients with GPC. This is not surprising considering the fact that rubbing alone can result in significant increases of tryptase in tears (Butrus et al., 1990). Eotaxin is not found to be elevated in the tears of GPC patients (Sarac et al., 2003). As in SAC and PAC, release of mediators from mast cells results in increased capillary permeability and inflammatory cell infiltration of eyelid tissue. Cytologic scrapings from the conjunctiva of patients with GPC exhibit an infiltrate containing lymphocytes, plasma cells, mast cells, eosinophils, and basophils. All of these factors contribute to discomfort and formation of the papillae. The differentiating pathophysiologic characteristics between GPC and VKC are important because they could be considered as possible clues to the differences in pathogenesis between these two ocular diseases.

3. Diagnosis

Examination of the underside of the upper eyelid will, in severe cases, reveal

large papillae with red, inflamed tissue. In milder cases of GPC, smaller papillae may occur. These papillae are thought to be caused by the contact lens riding high on the surface of the eye with each blink. In very mild cases, this tendency of the contact lens to ride up on the eye may contribute to the diagnosis in the absence of visible papillae. In cases of chronic GPC, tear deficiency may be a contributing factor. Redness of the upper eyelid on ocular examination is one of the earliest signs of GPC and this observation can facilitate early diagnosis. Abnormal thickening of the conjunctiva may progress to opacification as inflammatory cells enter the tissue.

4. Treatment

Reducing symptoms is the primary aim for management of GPC. A reduction in the wearing time of contacts from a few hours a day to total abstinence may be required. Once-a-day use contact lenses may be a consideration for persistent cases of GPC. However, in more serious cases, a more aggressive approach may be required to prevent ocular tissue damage. Over-the- counter “artificial” tears help to wash away environmental allergens and lens debris.

Topical mast cell stabilizers have proven effectiveness in the treatment of GPC (Meisler et al., 1982; Donshik et al., 1984; Sorkin and Ward, 1986; Allansmith, 1987). Dual acting drugs may be the best therapy for chronic GPC. A patient with GPC may require continued use of these drugs once they return to contact lens wear. Steroids have also been approved for the treatment of GPC (Asbell and Howes, 1997). Topical steroids may be used 4 times per day for 2–4 days (Bartlett et al., 1993). A return to contact lens wear can usually be accomplished, but may require a change in contact lens style or lens material.

B. Vernal Keratoconjunctivitis

VKC is a chronic, bilateral conjunctival inflammatory condition found in

individuals predisposed by their atopic background. An excellent review of the history and description of this disease was published by Buckley in 1988. Beigelman’s 1950 monograph “Vernal conjunctivitis” continues to be the most exhaustive compilation of this disease and is unmatched in current times. The list of easily recognized names in ophthalmology to have published regarding this entity is formidable: Arlt (1846), Desmarres (1855), von Graefe (1871), Axenfeld (1907), Trantas (1910), and Herbert (1903). In 2000, Bonini reviewed a series of 195 patients with VKC as the only allergic manifestation in 58.5% of patients (Bonini et al., 2000).

1. Clinical parameters

The onset of disease is generally before age 10; it lasts 2 to 10 years; and it usually resolves during late puberty. Only 11% of patients were greater than 20 years of age in the Bonini series (Bonini et al., 2000). Males predominate in the younger ages, but the M:F is nearly equal in the older patients. Young males in dry, hot climates are those primarily affected. The Mediterranean area and West Africa are areas of the greatest numbers of patients. It is relatively

unusual in most of North America and Western Europe. There is a significant history of other atopic manifestations such as eczema or asthma in 40–75% of patients with VKC. A family history of atopy is found in 40–60% of patients (Bonini et al., 2000). Seasonal exacerbation, as the name implies, is common, but patients may have symptoms year-round.

Severe itching and photophobia are the main symptoms. Associated foreign body sensation, ptosis, thick mucous discharge and blepharospasm also occur.

The signs are confined mostly to the conjunctiva and cornea; the skin of the lids and lid margin are relatively uninvolved compared to AKC. The conjunctiva develops a papillary response, principally of the limbus or upper tarsus. The tarsal papillae are discrete, greater than 1 mm in diameter, have flattened tops that may stain with fluorescein, and occur more frequently in European and North American patients (Buckley, 1988). Thick, ropy mucus tends to be associated with the tarsal papilla (Figure 11.4). These are the classic “cobblestone papillae”.

Limbal papillae tend to be gelatinous and confluent; and they occur more commonly in African and West Indian patients

(Buckley, 1988). Rao et al. (2004) recently described increased perilimbal pigmentation in an Indian population of patients with VKC. Horner–Trantas dots, which are collections of epithelial cells and eosinophils, may be found at any meridian around the limbus (Trantas, 1910). These changes may lead to superficial corneal neovascularization. The forniceal conjunctiva usually does not show foreshortening or symblepharon formation.

The corneal findings may be sight threatening. Buckley (1988) describes in detail the sequence of occurrence of corneal findings. Mediators from the inflamed tarsal conjunctiva cause a punctate epithelial keratitis. Coalescence of these areas leads to frank epithelial erosion, leaving Bowman’s membrane intact. If, at this point, inadequate or no treatment is rendered, a plaque containing fibrin and mucus deposits may develop over the epithelial defect (Rahi, 1985). Epithelial healing is then impaired, and new vessel growth is encouraged. This so-called shield ulcer (Figure 11.5) usually has its lower border in the upper half of the visual axis. With resolution, the ulcerated area leaves a subepithelial ring-like scar. The peripheral cornea may show a waxing

and waning, superficial stromal, gray–white deposition termed pseudogerontoxon. Iritis is not reported to occur in VKC.

2. Pathophysiology

Biopsy of a tarsal conjunctival papilla in VKC reveals distinct findings. The epithelium contains large numbers of mast cells and eosinophils, neither of which is found in normal individuals (Allansmith et al., 1979; Leonardi et al., 1995). Human mast cells may be categorized based on the presence of neutral proteases (Irani et al., 1986). The epithelium of VKC patients contains mast cells predominantly of the type containing the neutral proteases tryptase and chymase (Irani et al., 1990). Basophils are found in the epithelium, which may indicate one form of a delayed type hypersensitivity reaction is occurring. Leonardi et al. (1995) demonstrated eosinophils, neutrophils, and mononuclear cells in the hyperplastic epithelium. Brush cytology of the conjunctival epithelium from patients with VKC showed more eosinophils and neutrophils in patients with corneal erosion or ulcer than in those without (Miyoshi et al., 2001). Goblet cell density is not found to be elevated in the

conjunctival epithelium of VKC (Allansmith et al., 1981). Eosinophil major basic protein is deposited diffusely throughout the conjunctiva of VKC patients, including the epithelium (Trocme et al., 1989).

The substantia propria contains elevated numbers of mast cells compared to normal individuals (Allansmith et al., 1979; Leonardi et al., 1995). The predominant mast cell subtype found contains tryptase and chymase (Irani et al., 1990). Forty-six percent of the mast cells in the substantia propria contain fibroblast growth factor bFGF (Leonardi et al., 2000b). This may serve as a source of fibroblast growth and production of collagens. Eosinophil major basic protein granules are found close to mast cells in VKC (Trocme et al., 1989). As in the epithelium, the substantia propria contains increased numbers of eosinophils and basophils compared to normal tissue (Allansmith et al., 1979). A unique profile of lymphocytes is found. T-cell clones can be isolated from biopsy specimens of VKC tarsal conjunctiva. These CD4 T-cell clones show helper function for IgE synthesis in vitro and produce interleukin-4 (IL-4) (Romagnani, 1990). Calder et al. (1999), in a separate work, found IL-5 expressed in T-cell lines from vernal biopsy specimen. Cognate interaction with T-cells and the presence of IL-4 are needed for B-cell production of IgE (Romagnani, 1990). This would support the suggestion that IgE is produced locally. The substantia propria also has an increased amount of collagen. Fibroblasts from the tarsal conjunctival biopsy of VKC patients can be induced to proliferate by histamine and epithelium derived growth factor (Leonardi et al., 1995). Cyclosporin A, often used in VKC, has been shown in vitro to reduce collagen production and induce apoptosis of conjunctival fibroblasts from VKC patients (Leonardi et al., 2001).

The corneal epithelium of VKC patients has been shown to express ICAM-1, an important cell adhesion molecule (Temprano, 1995). Eosinophil peroxidase in contact with

human corneal epithelial cells causes disruption of cell adhesion (Hallberg, 1995). Eosinophil major basic protein and cationic protein are pro-inflammatory, and EMBP has been shown to be cytotoxic to corneal epithelium. In vitro, both of these damage the monolayers of human corneal epithelial cells, but not the stratified corneal epithelial cells in culture (Ward, 1995).

Specific IgE and IgG have been isolated from the tears of VKC patients (Sompolinsky, 1982; Ballow and Mendelson, 1980). Histamine (Abelson et al., 1980) and tryptase (Butrus et al., 1990) are elevated in the tears of VKC patients. The serum of VKC patients has been found to contain decreased levels of histaminase and increased levels of nerve growth factor (Mukhopadhyay et al., 1981; Bonini et al., 1999). In patients with VKC, 4 inflamma- tion-related factors, eotaxin, interleukin (IL)-11, monocyte chemoattractant protein (MCP)-1, and macrophage-colony stimulating factor (M-CSF), increased to 4 times the values in the control group. Seven inflam- mation-related factors, eotaxin-2, IL-4, IL- 6, interleukin-6 soluble receptor (IL-6sR), IL-7, macrophage inflammatory protein (MIP)-1 , and tissue inhibitor of metalloproteinases (TIMP)-2, increased to 8 times the control values (Shoji et al., 2006). Finally, VKC is reported to occur in patients with hyperimmunoglobulin E syndrome (Butrus et al., 1984).

3. Diagnosis

The diagnosis is relatively easily arrived at based on the history and presentation of findings. As indicated previously, VKC occurs predominantly in young boys living in warm climates. These patients have intense photophobia, ptosis, and the characteristic finding of giant papillae. The principal differential diagnostic entity is AKC. The two are compared and contrasted in Table 11.1. Tear fluid analysis and cytology, conjunctival scraping for cytology, and biopsy are rarely needed to assist in establishing the diagnosis.