Ruth van der Gaag

Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons

AIM To study the value of polymerase chain reaction (PCR) analysis, to detect viral DNA in recipient corneal buttons taken at the time of penetrating keratoplasty (PKP) in patients with an initial diagnosis of herpetic stromal keratitis (HSK). Since HSK has a tendency to recur, an accurate diagnosis of previous HSK could be the reason to start antiviral treatment immediately, thereby possibly decreasing the number of graft failures due to recurrent herpetic keratitis. METHODS Recipient corneal buttons and aqueous humour (AH) samples were obtained at the time of PKP from HSK patients (n=31) and from other patients (n=78). Eye bank corneas were also used (n=23). Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) infection were assessed by PCR and antibody detection. RESULTS The clinical diagnosis HSK could be confirmed by PCR for HSV-1 in 10/31 (32%). In these corneal buttons HSV-2 DNA was detected in 1/31 (3%) and VZV DNA in 6/31 (19%). Intraocular anti-HSV antibody production was detected in 9/28 AH samples tested (32%). In the other patient derived corneas HSV-1 DNA was detected in 13/78 (17%), including eight failed corneal grafts without clinically obvious herpetic keratitis in the medical history. In clear eye bank corneas HSV-1 was detected in 1/23 (4%). CONCLUSIONS PCR of HSV-1 on corneal buttons can be a useful diagnostic tool in addition to detection of intraocular anti-HSV antibody production. Furthermore, the results were suggestive for the involvement of corneal HSV infection during allograft failure of corneas without previous clinical characteristic signs of herpetic keratitis.

Macrophages play a role in the early phase of corneal allograft rejection in rats

Background. Rat corneal allograft rejection is delayed by repeated local injection of liposomes filled with clodronate (dichloromethylene diphosphonate), which selectively deplete macrophages. Various administration schedules of liposomes were tested to determine the optimum schedule for prevention of graft rejection. Cell subpopulations in the anterior segment of the eye were studied at different time points after transplantation to assess the kinetics of the immune response. Methods. AO rats were grafted orthotopically with corneal buttons from PVG rats. Postoperatively, rats remained untreated or received clodronate liposomes subconjunctivally. Clodronate liposomes were injected five times on postoperative days (PODs) 0, 2, 4, 6, and 8; or once, on POD 0 or 6; or twice on PODs 0 and 2 or PODs 0 and 6. Grafts were examined for signs of rejection clinically and immunohistologically. Results. All untreated rats rejected their grafts as did all five rats that received clodronate liposomes once on POD 6. In all the other administration schedules tested, graft survival was prolonged compared with the untreated control group (P <0.01). Injections of clodronate liposomes on PODs 0 and 2 proved to be the most effective treatment. Histologically reduced influx of virtually all cell types tested was found in this group. Conclusions. To prevent or delay graft rejection, it is necessary to administer clodronate liposomes in the early phase after corneal transplantation. These results suggest a role for macrophages in the afferent phase of corneal graft rejection.

Effect of local macrophage depletion on cellular immunity and tolerance evoked by corneal allografts

Purpose. To determine whether local macrophage depletion, via administration of clodronate liposomes, alters delayed type hypersensitivity (DTH) responses and induction of anterior chamber associated immune deviation (ACAID) after corneal allotransplantation. Methods. Clodronate liposome-treated and untreated rats received orthotopic corneal allografts and were tested for DTH responses and induction of ACAID towards donor antigens. Also in subconjunctivally treated and untreated rats, DTH responses were measured after subcutaneous immunization or induction of ACAID with allogeneic spleen cells. Results. Subconjunctival injected clodronate liposomes prevented grafted rats from developing donor-specific DTH as well as ACAID. By contrast, subconjunctivally injected clodronate liposomes had no effect on donor-specific DTH responses after systemic immunization or on the induction of ACAID with allogeneic cells. Conclusions. Depletion of macrophages at the time of corneal allografting seems to render the grafts immunologically invisible to the recipients. This could explain why these grafts survive “indefinitely” without any other form of therapy.

Autoimmunity against corneal antigens. I. Isolation of a soluble 54 Kd corneal epithelium antigen

Corneal epithelium antibodies were detected in patients with corneal melting disease and uveitis using an immunofluorescence technique with cryostat sections of corneas obtained from various species (man, guinea pig, rabbit, mouse, rat, cow, pig). No differences in results were found using these various substrates, indicating that the autoimmune response is directed against common non-species specific corneal epithelium antigens. The serum of a patient with corneal melting disease, containing a high antibody titer against corneal epithelium was used to identify and isolate one of the bovine corneal antigens. A 54,000 dalton protein was isolated, which was shown to be the major protein present in the corneal epithelium. Absorption studies with other tissues taken from human eyes showed that cornea epithelium, cornea devoid of epithelium, ciliary body and retina contained material which cross-reacted with the isolated bovine corneal epithelium antigen, whereas iris and sclera showed no detectable cross-reaction. The incidence of autoantibodies directed against this antigen was investigated in patients with corneal melting disease, corneal transplantion and in uveitis patients using an ELISA and comparing the results with those obtained with the immunofluorescence assay on rabbit cornea sections. A positive ELISA was always associated with a positive immunofluorescence test. The presence of antibodies against the 54 Kd antigen as detected by the ELISA could be confirmed by immunoblotting in 7 out of 9 positive sera tested. A large number of sera showed a positive immunofluorescence test but a negative ELISA against the 54 Kd corneal epithelium antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating cornea-specific antibodies in corneal disease and cornea transplantation

In order to establish the significance of circulating corneaspecific antibodies, we determined the presence of anti-corneal antibodies in the serum of 100 patients with corneal disease and in 50 healthy controls, and subsequently followed the pattern of antibody reactivity in 46 patients who underwent corneal transplantation. An indirect immunofluorescence test on cryostat sections of rabbit corneas was used for screening. The reactivity against two known bovine corneal epithelial proteins was also tested: a 54-kD protein (BCP 54) and an 11-kD protein (BCP 11/24). No significant difference in the presence and specificity of anti-corneal antibodies was observed between the group of patients with corneal disease, taken as a whole, and the healthy controls. Patients with keratoconus or non-immunological graft failure, however, were significantly more often positive for anti-corneal antibodies. Neither the presence of antibodies prior to corneal transplantation nor their appearance post-transplantation had a predictive value for corneal graft survival.

The influence of high molecular weight sodium hyaluronate (Healon®) on the production of migration inhibitory factor

The effect of sodium hyaluronate on the production of migration inhibitory factor (MIF) was studied in a two step MIF-assay. High molecular weight sodium hyaluronate (100 micrograms/ml), added during the inductory step of the MIF-assay, inhibited the production of MIF. The inhibitory effect did not appear to be due to physical factors such as steric hindrance, which may prevent mitogen binding, since cells preactivated with phytohemagglutinin A (PHA) did not produce MIF when incubated in the presence of sodium hyaluronate. The inhibitory effect was still measurable when the sodium hyaluronate was added upto two hours after stimulation of the mononuclear cells with PHA. Inhibition was also found when the cells were preincubated with sodium hyaluronate, and washed prior to mitogen stimulation. Sodium hyaluronate could only be removed from the cells by incubation with hyaluronidase or by incubation of the cells for at least two hours in culture medium, whereafter the cells could be stimulated to the same extent as normal untreated cells to produce MIF. This inhibitory effect on cytokine production may explain the reduced inflammatory reactions found both in vivo and in vitro in the presence of sodium hyaluronate.

Autoimmunity against corneal antigens. II. Accessibility of the 54 kD corneal antigen for circulating antibodies

Autoantibodies against corneal epithelium antigens are frequently found in patients with corneal diseases, but also in patients with uveitis. To investigate whether these autoantibodies play a primary role in the pathogenesis of corneal disease we studied the distribution of a 54 kD corneal antigen (isolated in a previous study) within the eye. Animal experiments were performed to determine the accessibility of this antigen by its corresponding antibody. Immunohistochemistry showed that the 54 kD corneal antigen was abundantly present in the rat corneal and conjunctival epithelium. A high concentration of this antigen was seen just above the nuclei in the basal cell layer of the corneal epithelium. The antigen could also be detected in the keratocytes of the corneal stroma, the corneal endothelium and the lens epithelium. The 54 kD corneal antigen was not detected in other parts of the eye, nor could it be found in the rat liver, kidney, spleen, lung or skeletal muscle. Incubation of intact rat eyes with antiserum against the 54 kD corneal antigen in vitro, resulted in a weak binding of immunoglobulins to the corneal surface epithelium cells. Passive transfer experiments, whereby rats received an intravenous injection of antiserum against the 54 kD corneal antigen resulted in a weak deposition of immunoglobulins in the corneal stroma and sclera. However, no antibodies were bound to the corneal epithelium. These observations show that although antibody production to corneal epithelium antigens is easily triggered, these antibodies do not reach the corneal epithelium.

Are cytokine patterns in aqueous humour useful in distinguishing corneal graft rejection from opacification due to herpetic stromal keratitis

Purpose: Intra-ocular cytokine profiles were determined to study the immunological mechanisms of corneal graft opacification due to rejection and/or herpetic stromal keratitis (HSK). Methods: Sera and aqueous humour (AH) were sampled shortly after the onset of corneal graft opacification, group I (n=18). In eyes with clear grafts, samples were taken 5 months after transplantation, group II (n=59). Samples of non-inflamed eyes, prior to cataract surgery, were used to determine baseline cytokine levels, group III (n=49). Total protein (TP) levels were measured with Bradford reagent and interleukin (IL)-6, IL-10, IL-4 and interferon (IFN)-γ with ELISAs. Results: All patients whos corneal grafts showed clinical evidence of graft opacification due to rejection and/or HSK were sampled. In the AH-samples of group I, increased levels of TP were found in 60% (9/15), IL-6 in 79% (11/14), IL-10 in 39% (7/18) and IL-4 in none (0/12). IFN-γ was detected in 19% (3/16), in the case of HSK only. In contrast, samples obtained from patients with clear grafts in group II showed increased levels of TP in 36% (20/55), IL-6 in 14% (8/57) and IL-10, IL-4 or lFN-γ in none (n=58). Conclusions: During corneal graft rejection and/or HSV-infection, increased levels of TP and IL-6 in AH confirmed anterior chamber inflammation with breakdown of the blood-aqueous barrier. Based on the data presented, cytokine patterns in the AH do not appear to distinguish corneal opacification due to graft rejection from that due to herpes keratitis.

Expression of the interleukin 1 receptor antagonist in the normal human cornea

The human cornea has been shown to express a number of inflammatory cytokines including IL-1, IL-6 and IL-8. In view of the potent proinflammatory activities of interleukin-1 (IL-1), regulatory mechanisms should be present in the human cornea to control IL-1 mediated inflammatory and immune responses. This is important for the maintenance of the integrity and transparency of the cornea. To test this hypothesis, the authors determined the presence of IL-1 receptor antagonist (IL-tra) in the normal human cornea using an enzyme-linked immunosorbent assay (ELISA). IL-tra is a natural antagonist of IL-1 and competes with IL-1 for the binding to its receptors thereby blocking the inflammatory response. Corneas were either tested immediately or after a 24-hour culture period. Furthermore, the authors separately analyzed the three layers of the cornea. Their results present evidence for the constitutive expression of the IL-tra protein in the normal human cornea and show that both epithelial and stromal cells produce IL-1ra. The epithelial cells are the major source of corneal IL-1ra immunoreactivity, and secrete IL-1ra during culture. Stromal cells contain detectable, albeit low amounts of cell associated IL-1ra. No IL-1ra was detected in the endothelial cell layer. A more accurate understanding of the balance between IL-1 and IL-1ra in ocular tissues and the role of the IL-1ra under physiologie and pathophysiologic conditions will be necessary for an eventual use of IL-1 receptor antagonist as a therapeutical tool.

Adhesion molecule expression in local-macrophage-depleted rats bearing orthotopic corneal allografts.

BackgroundRejection of corneal grafts is dependent on influx of T lymphocytes and macrophages. This process is partly regulated by adhesion molecules. Earlier investigations showed that corneal graft rejection in rats could be prevented by clodronate liposomes that selectively eliminate macrophages. In the present study the effect of macrophage depletion on adhesion molecule expression after corneal allotransplantation was investigated.MethodsOrthotopic corneal allografts were performed, after which rats received subconjunctival injections with clodronate liposomes or remained untreated. On various postoperative days, grafted rats were killed and mid-eye sections were stained for expression of ICAM-1 (CD54) and β2-integrins (CD18 and CD11b/c).ResultsIn the clodronate liposome-treated group grafts were not rejected, while in untreated rats grafts had a mean survival time of 12 days. During the first postoperative days a slightly enhanced expression of ICAM-1 in the conjunctiva and allografted cornea of clodronate liposome-treated recipients was seen. On day 12, however, ICAM-1 expression was markedly downregulated in the allografts of this treated group. The expression of β2-integrins was also significantly decreased in the allografts and recipient corneas of treated rats at this time point.ConclusionProlonged corneal graft survival in rats, obtained via local depletion of macrophages, correlates with diminished expression of adhesion molecules.