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Background: Ofloxacin (OFLX) is a fluoroquinolone-antibiotic with a broad antimicrobial spectrum that may have a potential role in the treatment of bacterial endophthalmitis. However, its elimination half life after intravitreal injection is short. To prolong the intravitreal antibacterial level OFLX was incorporated into liposomes. This study was performed to investigate the retinal toxicity of liposome-incorporated and free OFLX., Materials and Methods: OFLX was incorporated into multilamellar large vesicles. 0.1 ml of this suspension (= 180.2 microg OFLX) was injected into the midvitreous of rabbit eyes (n = 6). Free OFLX in doses of 100 microg, 500 microg and 1,000 microg was injected into the midvitreous of a second group of rabbit eyes (n = 18). The other eye served as a control and received empty liposomes or normal saline solution, respectively. Before injection and at the end of follow-up an ERG was obtained. After a follow-up of 1 day, 14 and 28 days the animals were perfused with glutaraldehyde and the eyes were examined by light- and transmission electron microscopy., Results: The ERG as well as the histologic studies did not reveal any pathological changes after injection of liposome-incorporated OFLX compared to the control eyes. Significant reduction of the ERG was observed after 500 microg free OFLX in 2 out of 6 eyes after 1 and 14 days, respectively, and in 2 eyes 1 day after 1,000 microg free OFLX. Three days after injection of 1,000 microg OFLX the retina showed focal destruction in 1 out of 6 eyes. In another eye with the same dose 14 days after injection the photoreceptor outer segments showed disorganisation., Conclusion: This study shows that liposome-incorporated OFLX did not have any retinal toxicity in this animal model. Free OFLX appears to have no retinal toxicity in rabbit eyes at a dose of 100 microg after intravitreal injection. Injection of higher doses resulted in ERG changes and marked retinal damage.

Corneal allograft rejection culminates in a series of interactions between different classes of antigen presenting cells, cytokines and leukocytes. Tumor necrosis factor-alpha (TNF-alpha) was recently reported to be elevated in acute rejection of solid organ transplants. This cytokine is released early in immune activation and may be detected in the peripheral circulation. Serial determinations of TNF-alpha serum levels were performed following experimental corneal allografts. Lewis rats received 3.5 mm orthotopic corneal grafts of MHC-incompatible Wistar-Furth donors. TNF-alpha concentrations were measured in serum samples collected pre- and postoperatively and measured by micro ELISA. Clinical observations revealed graft rejection in 65.5% of corneal transplants 14 +/- 4 days following grafting. The mean serum level of TNF-alpha in control animals without corneal graft (group I) was 41 +/- 12 pg/ml. Animals following keratoplasty without allograft rejection (group II) showed a mean TNF-alpha level of 54 +/- 16 pg/ml that did not differ from group I. The rejection group III displayed significantly higher TNF-alpha levels (98 +/- 16 pg/ml, p < 0.05). These significantly elevated levels were found even before the diagnosis of rejection was established by clinical criteria. These data suggest systemic immunoreactivity to corneal allografts. Elevated levels of cytokines may provide valuable information in recipients undergoing rejection and may also provide a rationale for systemic immunotherapy in some instances.

The outer surface of the eye is constantly exposed to a wide array of microorganisms. To protect the integrity or the ocular surface and to retain corneal transparency, a number of defense mechanisms have evolved. This article discusses the host mechanisms of the eyelids-, tears, cornea and conjunctiva. These host defense mechanisms are identified as either a native, nonspecific defense or a specifically acquired immunological defense requiring previous exposure to an antigen and the development of specific immunity. Nonspecific components that protect the eye include the eyelids, ocular surface epithelium, normal flora and tear proteins. Specifically acquired immunity in tears, cornea and conjunctiva involves the interaction of antigen-presenting cells, lymphocytes and humoral components of the immune system.

Unlabelled: Inflammatory stimuli induce the expression of adhesion molecules on leukocytes and endothelial cells. The adhesive forces between leukocytes and the vascular endothelium are antagonized by the shear stress of the flowing blood. The aim of this study was to examine how differences in the apparent wall shear rate measured in iris venules with varying caliber affect leukocyte-endothelium interaction., Methods: The microvasculature of the iris in Lewis rats (n = 6) was examined using intravital fluorescence microscopy [2]. Blood flow was visualized with FITC-HES and leukocytes were stained with rhodamine 6G. Venules (n = 110) ranging in diameter from 20-60 microns were analysed with regard to the following parameters: Flux of leukocytes, velocity of leukocytes in the center stream, number of rolling and adherent leukocytes. The apparent wall shear rate was calculated from the center stream velocity and the vessel diameter. Animals were examined 4 h after administration of endotoxin from S. typhimurium (500 mg/kg body wt i.p.), when leukocyte-endothelium interaction is strongly enhanced., Results: The flux of leukocytes, the absolute number of rolling leukocytes and the velocity of leukocytes in the center stream was higher in larger than in smaller venules. The apparent wall shear rate decreased with increasing vessel diameter. Calculated values were 806 +/- 59 s-1 for vessels 20-30 microns in diameter and 483 +/- 34 s-1 for vessels of 50-60 microns (mean +/- SEM). The number of adherent leukocytes per mm2 endothelial surface and the rolling fraction did not show significant differences between vessels with varying caliber., Conclusion: Although the shear rate declined with increasing vessel diameter, we could not observe an increase in rolling and adherent leukocytes. The effects of vessel diameter on leukocyte adhesion to the vascular endothelium in postcapillary iris venules are minimal.

Background: Traumatic wound dehiscence after penetrating keratoplasty is probably underestimated., Patients: From the files of the University Eye Hospital Tübingen (1981-1993), 15 patients with a traumatic wound dehiscence after penetrating keratoplasty were investigated., Results: Latency between corneal grafting and wound rupture ranged from 1 month to 25 years (mean 6.2 years). Wound dehiscence was quite evenly distributed over the circle. Intraocular tissue was lost to a variable extent. Three eyes were primarily or secondarily enucleated. Seven eyes (47%) with an originally reduced but useful vision became blind or were removed. Most (67%) of the resutured grafts lost transparency., Conclusions: Traumatic wound dehiscence is a serious and not very rare complication after penetrating keratoplasty. Functional results are poor.

Purpose: Liposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS., Methods: 12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacyl-phosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion., Results: The CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small., Conclusions: Our results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposome-encapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes.

We report clinical experiences with six patients treated at the University of Tübingen between 1982 and 1994 for symptoms of typical and atypical ocular manifestations of Cogan's syndrome. All cases had systemic symptoms in addition to involvement of the cochleovestibular system. Otologic symptoms were the initial presentation in four cases. Vestibular dysfunction often preceded hearing loss. Five patients had keratitis and also demonstrated signs of ocular inflammation diagnosed as scleritis or episcleritis. In two patients these ocular symptoms were the first signs of Cogan's syndrome, recurred periodically and did not respond to corticosteroids. Three patients were diagnosed early, allowing corticosteroid treatment to begin early with stabilization of hearing. Total bilateral deafness could not be prevented in the remaining patients. The importance of being aware of auditory-vestibular dysfunction occurring in patients with ocular inflammation and the role of early immunotherapy in preventing deafness has to be emphasized.

In skin-blistering diseases, alteration of cellular adhesion results in a loss of cohesion of the epithelium of the skin and the mucous membranes. These disorders are often genetically determined and involve highly specific autoantibodies. A recent workshop discussed the immunology of these diseases.

Behçet's disease (BD) is a multisystem vasculitis of unknown origin. In this retrospective study we analyzed the therapy of 32 patients seen between 1978 and 1993 at the Departments of Rheumatology, Ophthalmology, and Dermatology of the Tübingen University Clinic. The aim of this study was to evaluate the efficacy of different therapeutic strategies concerning different organ manifestations of the disease, especially eye disease. A total of 20 patients had cutaneous manifestations or arthritis. Whereas treatment with colchicine (Col), azathioprine (AZA), cyclosporine (CSA), or steroids (Ster) produced only partial remissions, a combination of CSA, AZA, and steroids led to complete remissions. Interferon-gamma (IFN-gamma) therapy led to remission rates of 60% (complete) and 20% (partial). In all, 22 patients had uveitis (posterior or panuveitis). Steroids were effective in only 50% of the patients and Col was partially effective in 66%. AZA induced a remission in 71% of cases and CSA was partial effective in 60%. The threshold combination of AZA, CSA, and Ster induced a complete remission in 66% of the patients. IFN-gamma was ineffective in 80%. IFN-alpha was used in one patient only and induced a complete remission. These results demonstrate that although our patient group is too small to allow significant conclusions to be drawn, in terms of the literature, for mucocutaneous disease and arthritis, IFNs might be the best therapy, whereas for uveitis as well as other more severe features of the disease, CSA or AZA + Ster should be used. If the latter are ineffective, the threefold combination (AZA, CSA, Ster) is probably the most effective alternative. The significance of IFN-alpha will be evaluated in further studies.

Noninfectious ulceration of the peripheral cornea remains a major diagnostic and therapeutic challenge. The pathogenesis in most of these disorders is unclear, however, on the basis of systemic connective tissue diseases, autoimmune mechanisms are most likely involved. The peripheral cornea has distinct morphological and immunological characteristics that predispose for inflammatory reactions. Major differences exist regarding humoral and cellular components of the immune system. In the peripheral cornea there is more high-molecular IgM and initial complement component C1 than in the central cornea and may predispose for immune complex formation. The close contact to the conjunctival vasculature provides the basis necessary to generate an immune response. Langerhans cells and macrophages as important antigen presenting and processing cells are present in higher number in the peripheral cornea. Autoimmune diseases that affect the peripheral cornea include collagen vascular diseases and Mooren's ulcer. Although this association is obvious in advanced rheumatoid arthritis more subtle forms of polyarteritis nodosa or systemic lupus erythematosus require careful medical evaluation and workup. Ocular manifestations may present as the initial clinical signs and require careful workup in these potentially lethal disorders.

Unlabelled: Erythema exudativum multiforme major (EEMM), also know as Stevens-Johnson syndrome, may cause severe conjunctival and corneal alterations. The etiology remains unknown. The aim of are study was to evaluate the clinical course, therapy and prognosis of ocular involvement., Patients and Methods: In a retrospective study, we evaluated ten patients with EEMM who were treated between 1986 and 1994 at the University Eye Clinic Tübingen., Results: The age of the four female and six male patients varied between 5 and 70 years. Drugs as a possible precipitating factor were found in all cases (acetylsalicylicacid, sulfonamide, erythromycin, cotrimoxazole, Valproinicacid, paracetamol). Eight patients experienced an infection at the same time. Clinically, we were able to differentiate two groups regarding severity of the disease. Seven patients were characterized by ocular alterations that only involved the conjunctiva, leading to conjunctival scars. In those cases local treatment with antibiotics and corticosteroids was effective. In the second group severe ocular damage occurred, with symblephara, keratinization and consecutive perforating keratoplasty. Secondary infections became evident in a total of six cases from the two groups., Summary: Drugs as a possible precipitating factor could be identified in all cases. In eight of ten patients previous infection was known. This agrees with literatures reports of viral infections (e.g. herpes simplex) as cofactors causing EEMM, especially for the aggressive form, remains unresolved. The danger of possible superinfection should always be taken into consideration.

Pemphigus vulgaris (PV) is an immune-mediated vesiculobullous disease of the skin and mucous membranes. Generally, patients with PV present first with oral lesions, which may precede the cutaneous lesions, such as bullae and erosions, by several months. An ocular manifestation is unusual. The most common ophthalmologic involvement in pemphigus is conjunctivitis, but without progressive scarring such as occurs in ocular cictricial pemphigoid. Corneal involvement is very rare. CASE REPORT. We report on severe ocular involvement in a 56-year-old male Turkish patient with PV. Ophthalmologic findings included conjunctival cicatrization, corneal ulceration and corneal perforation, despite immunosuppressive therapy with azathioprine and corticosteroids. A penetrating keratoplasty was performed, but rapidly failed because of corneal vascularization. CONCLUSION. If a chronic bullous dermatosis leads to severe eye involvement, PV should be considered in the differential diagnosis besides ocular cicatricial pemphigoid. Pemphigus vulgaris can be differentiated on the basis of clinical findings and histopathological and immunohistological features. Generally, PV can be treated with steroids or with a combination of an immunosuppressant and steroids. However, the combination of prednisone and azathioprine could not prevent corneal perforation in our patient.

Background and Objective: An inflammatory response produced by excimer laser photorefractive keratectomy (PRK) may be associated with the subsequent corneal haze and regressions in refractive error observed after treatment. Complement-derived anaphylatoxins, potent mediators of inflammation, may have a role in postoperative healing., Materials and Methods: Twenty right human donor corneas underwent a 6-D excimer laser PRK treatment. The corresponding left donor corneas served as the controls. After incubation in tissue culture media for 6 hours and elution in phosphate-buffered saline with EDTA for 24 hours, complement-derived anaphylatoxins C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay., Results: Compared with control corneas, the excimer PRK corneas failed to demonstrate a significant increase in C3a, C4a, or C5a levels (P > .05)., Conclusions: These results suggest that the excimer laser at this dose does not activate significant complement in the cornea.

Purpose: To analyze leukocyte-endothelium interaction in iris venules of living rats and to quantify changes of leukocyte dynamics in endotoxin-induced uveitis (EIU)., Methods: Lewis rats received an intraperitoneal injection of 100 micrograms of lipopolysaccharide (LPS; Salmonella typhimurium). Using intravital fluorescence microscopy, the iris vessels were examined, 2, 4, 6, 10, 14, 24, and 72 hours after LPS injection. A setup for intravital fluorescence microscopy of iris venules in the rat is described. Images are recorded with a video camera and stored on S-VHS videotape for off-line analysis. For contrast enhancement, erythrocytes and plasma were stained with fluorescein isothiocyanate (FITC) and FITC-hydroxyethylstarch, respectively. Rhodamine 6G was used for intravital staining of leukocytes. Resolution and magnification (x850) of the system facilitates observation of individual cells in the bloodstream in real time. Leukocytes were either flowing in the center stream, rolling along the endothelium, or firmly adherent. Image analysis provided data on microvascular leukocyte flux and leukocyte velocity., Results: The percentage of leukocytes rolling on postcapillary venular endothelium increased significantly (P < 0.05) 4 hours after endotoxin administration, as did the number of firmly adherent cells. Leukocyte-endothelium interaction reached its maximum 6 to 10 hours before an increase of inflammatory cells in the aqueous humor. The response to endotoxin was reversible, subsiding to near-normal values after 72 hours., Conclusions: Intravital fluorescence microscopy provides data on microvascular parameters, including the number of rolling and sticking leukocytes on vascular endothelium. Inflammation of the anterior uvea was characterized with regard to leukocyte recruitment from blood to the vessel wall.

Amphotericin B remains an important antifungal agent in the treatment of ocular mycosis. Since topical ocular application is limited because of ocular irritation and poor penetration, we studied the pharmacokinetics of amphotericin B encapsulated in unilamellar liposomes (AmBisome). One drop (20 microliters) of AmBisome or an equivalent concentration of amphotericin B was applied to rabbit eyes. Drug concentrations were measured 15, 60, 120 and 240 min following administration of the agents by HPLC in cornea and aqueous humor. The effect of intact (group A) and debrided corneal epithelium (group B) was also studied. Corneal amphotericin B levels were significantly higher (P < 0.01) after 15 min in animals receiving amphotericin B as compared to AmBisome in group A. At later time points no differences in the corneal drug levels were found, and the drug levels following AmBisome application were remarkably stable. Epithelial removal resulted in increased corneal drug levels following application of both amphotericin B preparations. Significantly higher drug levels were observed after free amphotericin B treatment at 15-60 min (P < 0.01). Drug levels in the aqueous humor did not differ between the two amphotericin B preparations and remained below therapeutically effective concentrations. These results suggest that topically delivered AmBisome provides stable corneal drug levels, but has the potential benefit of lowered ocular toxicity.

Between 1980 and 1992 we performed 128 keratoplasties à chaud on 69 (54%) eyes with progressive bacterial and on 59 (46%) eyes with progressive herpetic keratitis. In 44 of the eyes the corneas were spontaneously perforated, and in 28 (22%) a descemetocele was present. Only grafts that had been followed for at least 1 year (3.2 +/- 2.4 years) were analyzed. During this time 88 (95%) of the severely threatened eyes were preserved. In the operated eyes visual acuity improved in 20 (54%) in the bacterial and in 36 (69%) in the herpetic group. The main complications were immunological allograft rejection, cataract progression and recurrence of herpes. Allograft rejection occurred in 29 (71%) of the bacterial grafts and remained irreversible in 18 (44%) cases. 30 (63%) eyes in the group with herpetic corneal lesions developed graft rejection, 19 (37%) of these rejections were irreversible. Cataract occurred in the postoperative course in 25 (61%) of the bacterial eyes and in 23 (44%) of the herpetic ones. Recurrence of herpes presented a specific problem in this group and appeared in 12 (23%) of the grafts during a follow-up of 1 month to 12 years (3.8 +/- 2.4 years). Our results demonstrate that keratoplasty à chaud is a valuable form of managing urgent pathological processes of the cornea and shortening the course of the disease. This procedure also allows satisfactory primary functional results in many cases, however, secondary re-keratoplasty and cataract surgery are necessary to achieve functional rehabilitation.

Background: Cogan's syndrome is an uncommon disease characterized by ocular inflammation, vestibuloauditory dysfunction and symptoms of systemic disease. The etiology is unknown, however there is evidence for an autoimmune pathogenesis. The "typical" Cogan's syndrome presents as bilateral interstitial keratitis and progressing vestibuloauditory dysfunction. The presence of other inflammatory manifestations in addition of keratitis has been termed as "atypical" Cogan's syndrome., Patients: We report on six patients presenting with typical as well as atypical ocular manifestations between 1982 and 1994. Typically, the illness was accompanied by systemic symptoms. Each patient had audiovestibular involvement, that was the initial presentation in 4 cases. Vestibular dysfunction often preceded hearing loss. Five of our patients not only presented with keratitis but also demonstrated signs of ocular inflammation diagnosed as scleritis or episcleritis. In two patients these ocular symptoms were the first signs of Cogan's syndrome, recurred periodically and did not respond to corticosteroids., Results: In 3 patients that were diagnosed early and treatment with corticosteroids was initiated early, hearing could be stabilized, in the remaining patients total bilateral deafness could not be prevented., Conclusion: The importance of being aware that vestibuloauditory dysfunction may occur in patients with ocular inflammation, and the fact that early immunotherapy may prevent the risk of deafness, has to be emphasized.

Behçet's disease (BD) is a systemic disease of unknown etiology, characterized by multiple organ involvement. Although its incidence is high in the Mediterranean countries and in Japan, BD is not often found in German patients. We therefore compared the epidemiological and clinical involvement of BD in 39 patients of German and of Mediterranean origin. The first manifestation was oral aphthosis in 72% of patients, followed by eye involvement (62%), skin lesions (25%), and arthritic problems (23%). Eventually, all our patients developed oral aphthosis. Whereas Germans had eye involvement in only 55% of cases (Mediterraneans, 71%), they came up with venous thrombosis or thrombophlebitis more often (64%) as compared with Mediterraneans (29%). The first ocular symptom was anterior uveitis in 87.5% of patients; the visual prognosis was poor due to occlusive retinopathy. Among 22 eyes of 12 patients, 16 had a visual acuity of 0.1 or less after 5 years of disease. We found a similar association with human leukocyte antigen B5 (HLA-B5) in the German patients (66.7%) and in the Mediterranean group (54.4%). There was no significantly higher HLA-B5 association with the uveitis or arthritis type. These results demonstrate a few differences between the two ethnic groups but illustrate that in Germans the disease also has a poor prognosis.

Transcorneal drug transport is normally limited by the intrinsic permeation characteristics of the corneal epithelium. However, liposomes, i.e., phospholipid vesicles composed of phospholipid membranes, have recently attracted attention as carriers of topically applied agents. The present study therefore describes a qualitative and quantitative laboratory investigation of interactions between corneal epithelial cells and liposomes. The lipid bilayers and interior spaces of liposomes were labelled with different fluorophores. Fluorescence microscopy revealed a rapid uptake of rhodamine B-labelled liposome bilayer components by the epithelial cell membrane and the cytoplasm. Simultaneously, intracellular uptake of aqueous liposome content was indicated by uniform fluorescence of the cytoplasm due to carboxyfluorescein (CF). The fluorimetric experiments showed that the uptake of liposomes by SIRC cells depended on liposome concentrations and the time of exposure of the cells to the liposomes, and that saturation effect characteristics were present. Cell fluorescence dropped by approximately 45% when the incubation temperature of the cells was reduced from 37 degrees C to 4 degrees C. Both this phenomenon and a significant reduction in liposome uptake (p < 0.05 and p < 0.01) after incubation with the metabolic inhibitors 2-deoxyglucose and sodium azide indicated active, energy-dependent processes. Phagocytosis in cell-liposome interactions was directly shown by a significant reduction in cell fluorescence (p < 0.05) after application of the actin inhibitor cytochalasin B. The results presented here give concrete data on interactions between liposomes and superficial cells of the eye in vitro.

We present a case of ophthalmomyiasis externa in a 54-year-old man in southwest Germany (Baden-Württemberg). After the patient had been attacked by a fly, he complained of a foreign-body sensation and itching in his right eye and developed purulent conjunctivitis. Slit-lamp examination revealed the presence of translucent, rapidly moving insect larvae in the conjunctival sac and on the corneal surface. Following removal of all larvae and instillation of gentamicin, the symptoms completely resolved within 2 days. Two of the nine larvae removed were examined by light microscopy and taxonomically classified as first-instar larvae of the sheep botfly Oestrus ovis L. 1758. Although this fly is the most common causative agent of external ophthalmomyiasis in the world, infestations in central Europe have only rarely been reported. Besides the case report and a detailed description of the larvae, an overview of the literature on European cases of ophthalmomyiasis externa caused by Oestrus ovis is given.

Purpose/methods: We studied a rare initial manifestation of myelodysplastic syndrome in an 82-year-old woman who had acute secondary glaucoma in the right eye and mature cataracts in both eyes., Results/conclusion: Therapy with glaucoma control medications and cataract extraction in the right eye resulted in expulsive hemorrhage and subsequent enucleation of the right eye. After cataract extraction, examination of the left eye disclosed a central serous retinal detachment and hemorrhage. Histopathologic analysis of the right eye demonstrated myelocytic and lymphocytic infiltration.

Purpose: Monoclonal antibodies (mAb) have generated interest as therapeutic agents. Limited data are available on the treatment of corneal graft rejection. The purpose of this study was to assess the use of topically applied mAb on experimental corneal grafts., Methods: W 3/25, an IgG 1 mouse antirat mAb that recognizes a CD4+ cell subset, was used to treat Lewis recipient rats that received orthotopic corneal grafts of Wistar-Furth donors. Recipients were randomly assigned to receive topically applied drops of liposome-incorporated anti-CD4 mAb (LIP-anti-CD4 mAb), an equivalent amount of free anti-CD4 mAb, an isotype-matched control mAb encapsulated in liposomes (LIP-control mAb), or empty liposomes (emp-LIP) 5 times daily for 10 days. To investigate the immunologic effect of mAb treatment, flow cytometry of the targeted cells and cytotoxic activity of lymphocytes were analyzed., Results: Application of LIP-anti-CD4 mAb was effective in reducing the rejection rate (P < .05) and in prolonging the mean survival time of corneal grafts that underwent rejection (P < .05). In contrast, no significant effect on graft outcome was observed after the application of control agents. Flow cytometry analysis did not reveal systemic depletion of the targeted lymphocyte subset in any anti-CD4 mAb treated animals. Rejected grafts elicited a cellular cytotoxic immune response in a cell-mediated lymphocytotoxic assay independent of the treatment given., Conclusion: The results suggest that treatment with topically applied LIP-anti-CD4 mAb prolongs graft survival in orthotopic corneal grafts in a rat model. The beneficial effect of LIP-anti-CD4 mAb, probably due to enhanced intraocular delivery, was achieved by using relatively low doses of mAb.

Experimental models of autoimmune uveitis are consistently associated with pinealitis. To investigate the interaction between the neuroendocrine and immune systems in humans with uveitis, we measured serum levels of the predominant pineal hormone melatonin (MEL), prolactin (PRL) and interleukin-2 (IL-2). A total of 100 patients with different forms of uveitis and 30 age-matched, healthy blood donors were evaluated retrospectively. The day-time MEL was reduced significantly (P < or = 0.01) in patients with iritis and iridocyclitis, and highly significantly (P < or = 0.001) in patients with intermediate uveitis, chorioretinitis and panuveitis. In 38% of patients day-time MEL levels in plasma were below the limit of detection. PRL was significantly reduced (P < 0.01) in patients with intermediate uveitis. IL-2 was reduced to about 50% of control values in all groups of patients. The results suggest a possible neuroendocrine-immune interaction in uveitis patients.

We investigated the ability of liposomes to deliver the immunosuppressive agent cyclosporine A (CsA) to the cornea, anterior sclera, aqueous humor, and vitreous in rabbit eyes. One drop (10 microliters) of liposome-encapsulated CsA (CsA-LIP) or olive oil drops containing an equivalent concentration of CsA (CsA-DR) were administered at 15-minute intervals within the first hour and then one hourly over a 6-hour period. In addition, collagen shields soaked for 30 minutes in the liposome preparation (CsA-LIP-CS) were tested in vitro and in vivo as a new drug delivery approach. CsA levels were measured by fluorescence-immunoassay after 1, 3, or 6 hours of drug administration. CsA levels in this study were highest in cornea and anterior sclera. In animals receiving either CsA-DR or CsA-LIP, CsA levels generally increased from 1 to 6 hours. In animals receiving a single application of CsA-CS-LIP, CsA levels peaked at 3 hours and declined at 6 hours in cornea and sclera. CsA-LIP and CsA-CS-LIP delivered significantly higher levels of CsA to the cornea and sclera at 1 and 3 hours than CsA-DR. In aqueous and vitreous humor, CsA levels increased from 1 to 6 hours in animals receiving either CsA-DR or CsA-LIP. On the other hand, animals receiving a single application of CsA-CS-LIP had lower levels of CsA at 6 hours than at the earlier time points. Animals receiving CsA-LIP or CsA-CS-LIP had significantly higher levels of CsA in aqueous and vitreous humor at 1, 3, and 6 hours than animals receiving CsA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the ability of collagen shields impregnated with gentamicin sulfate and dexamethasone to deliver medication into rabbit eyes. We compared the aqueous humor gentamicin and dexamethasone levels delivered by collagen shield and subconjunctival injection therapy at five time points over a ten-hour period, by using a fluorescence polarization immunoassay and high-pressure liquid chromatography. Our in vitro studies showed that impregnated collagen shields released most of the gentamicin within the first 30 minutes of elution, whereas dexamethasone was released gradually over a ten-hour period. The collagen shields impregnated with gentamicin and dexamethasone produced aqueous gentamicin levels that were significantly lower (P = .014) than those produced by subconjunctival injection therapy at 30 minutes and that were comparable to subconjunctival injection therapy at the other time points. Minimum inhibitory concentrations of gentamicin (approximately 4 micrograms) were observed in aqueous humor within 30 minutes in rabbits that received subconjunctival injections and at one hour in rabbits treated with impregnated collagen shields. The impregnated collagen shields produced aqueous dexamethasone levels that were significantly lower (P = .004) than those produced by subconjunctival injection therapy at one hour, significantly higher (P = .028) than subconjunctival injection therapy at six hours, and comparable to subconjunctival injection therapy at the other time points. Collagen shield delivery of gentamicin-dexamethasone may be comparable to subconjunctival injections and provide an alternative therapy after intraocular surgery.

Purpose: To investigate the use of topically applied FK506, a new immunosuppressive compound, systemic and ocular absorption was determined in serum and various ocular tissues., Methods: Two drops of 20 microliters FK506 were applied using oil dissolved (OD-FK506) or liposome-bound (LIP-FK506) drug. FK506 concentrations were measured at intervals of 30, 60, and 120 minutes by immunoassay., Results: After application of OD-FK506, the highest concentrations of FK506 were found in the cornea and the conjunctiva (200-1200 ng/g) with substantial drug also present in anterior and posterior sclera. Relatively low concentrations were measured in the aqueous and vitreous humors (0.2-1.0 ng/g) of these animals. Using the same treatment regimen, LIP-FK506 was effective in delivering significantly higher drug concentrations (P < 0.05) to all ocular tissues and particularly aqueous humor (5-28 ng/g) and vitreous humor (12-22 ng/g) at all time points. During the observation period drug concentrations produced by LIP-FK506 remained well above the therapeutic range. FK506 levels were not detectable in serum (< 0.2 ng/ml) with either drug formulation., Conclusion: These findings indicate that liposomes may be a promising formulation for topical use of FK506 in ocular immune-mediated diseases.

Purpose: To study the effects of different formulations of topical cyclosporine (Cyclosporin A [CsA]) on corneal allograft rejection in a rat model., Methods: Female Lewis rats received penetrating keratoplasties from female Wistar-Furth donors. A total of 78 allogeneic grafts were performed. An additional 15 syngeneic grafts (Lewis) were used as technical controls. Two CsA preparations with equivalent drug concentrations (2.1 mg/ml) were applied as drops: CsA encapsulated in large unilamellar liposomes (CsA-LIP) and CsA dissolved in olive oil (CsA-DR). Allogeneic grafts were randomly assigned to receive CsA-LIP or CsA-DR beginning on the day of surgery five times daily for 10 days. Animals without any treatment or receiving empty liposomes (EM-LIP) were used as treatment controls. Grafts were graded three times weekly and a rejection index was generated based on graft clarity, neovascularization, and vessel size., Results: All syngeneic grafts remained clear over the observation period of 60 days. Rejected allogeneic grafts without any treatment and those receiving EM-LIP or CsA-DR showed a mean survival time (+/- standard deviation) of 14 +/- 4, 14 +/- 5, and 14 +/- 4 days, respectively. There was no significant difference in mean survival time between the grafts without any treatment and those in CsA-DR or EM-LIP treatment groups. The mean survival time of rejected grafts in animals receiving CsA-LIP was prolonged to 20 +/- 4 days. There was a significant difference in the mean survival time between the CsA-LIP treatment group and groups receiving CsA-DR, EM-LIP, or no treatment (P < or = 0.05). The Kaplan-Meier survival curve of the CsA-LIP treatment group was significantly different from the other experimental groups. The graft survival rate in the CsA-LIP group was 77%, whereas the rate was 37% in the non-treated group, 45% in the CsA-DR group, and 36% in the EM-LIP group., Conclusion: Encapsulation of CsA in liposomes might be a promising formulation for use in the prevention of corneal graft rejection.

Over a 5-year period, a consecutive series of 52 eyes in 46 patients with uveitis underwent extracapsular cataract extraction. Twenty-eight of these cases received a posterior chamber intraocular lens (IOL). During an average follow-up of 25 months (range 7 to 58 months) 71% of eyes receiving an IOL achieved postoperatively a visual acuity of 0.5 or better; 54% of aphakic eyes reached this level. Persistent cystoid macular edema limited the visual improvement to 20/200 in 6 patients; none of the patients developed cystoid macular edema postoperatively on clinical observation. Intraocular hypertension occurred postoperatively in 12 eyes, but was limited to a 4-week postoperative period in 9 cases. YAG laser capsulotomy was performed in 2 eyes with opacification of the posterior lens capsule without any further complications. The results suggest that uveitis patients benefit from cataract extraction and in selected cases can tolerate IOL implantation without major complications.

Although Staphylococcus epidermidis is the most common cause of postoperative pseudophakic endophthalmitis, little is known about the immune response to S. epidermidis-induced endophthalmitis. Using a rabbit model, the immune response to an intravitreal injection of 7000 S. epidermidis (group 1) or 30,000 S. epidermidis (group 2) organisms was investigated. Clinical evaluations showed that rabbits in group 2 had a more severe inflammatory reaction in the conjunctiva, cornea, iris, and vitreous than those in group 1. The inflammatory reaction in group 1 largely resolved by day 30; group 2 continued to show a severe inflammatory response. Histopathologic findings correlated with clinical findings, with rabbits in group 2 showing a more severe inflammatory reaction in both the anterior and posterior segments of the globe. Positive vitreous cultures for S. epidermidis were present in rabbits in group 1 on days 3, 7, 10, 14, and 21 but not thereafter. However, group 2 had higher vitreous colony counts at days 3, 7, and 14 and negative vitreous cultures thereafter. Neither group showed delayed hypersensitivity to S. epidermidis antigens (evaluated by skin tests). Serum immunoglobulin (Ig) G antibody levels to phenol-inactivated S. epidermidis and glycerol teichoic acid (GTA) increased progressively, reached a peak at days 10-14, and then declined in both groups. Serum IgA antibody levels to these antigens were not detected. Group 2 had a more prolonged IgG antibody response in vitreous and aqueous than group 1. Tear fluid showed the weakest IgG and IgA antibody response to S. epidermidis and GTA. S. epidermidis-induced endophthalmitis was associated with a humoral but not a delayed hypersensitivity response to this organism.

The authors examined the effect of systemic administration of cobra venom factor (CVF) on hemolytic complement levels in guinea pig sera and corneas. Guinea pigs received repeated intraperitoneal injections of CVF. Sera and corneas were obtained before and 1, 2, 5, 8, 10, 12, 16, and 20 d after the initial injection of CVF. Total hemolytic complement activity was measured by lysis of sheep erythrocytes sensitized with rabbit antibodies. There was a significant decrease in serum complement levels at days 1, 2, 5, 8, and 10 after the initial injection of CVF. After this, complement levels rose to levels that exceeded preinjection values. Complement levels in corneas also declined after CVF injections with a significant decrease at days 2, 5, 8, and 10 and a return to normal thereafter. Corneal complement depletion and restoration lagged behind serum. Moreover, the rate of complement depletion and restoration was greater for serum than cornea. Light microscopic examination of representative corneas at each time did not show local tissue changes during decomplementation. This is first study (to the authors' knowledge) to document corneal complement depletion by systemic administration of CVF. Low-to-absent corneal complement levels were present 2 d after the initial injection of CVF and persisted for 6 d. This model could be used to study the influence of complement on traumatic and immunologic injuries to the cornea.

We evaluated the effect of collagen shields presoaked with amphotericin B on the treatment of experimental Candida albicans-induced keratitis. Treatment results were compared to those of amphotericin B eyedrops instilled hourly. Forty-eight albino rabbits received intrastromal injections of 10(8) C. albicans organisms. Twenty-four hours later, eyes were treated for eight hours each day with hourly instillation of 0.15% amphotericin B drops, hourly instillation of saline drops, or application of a collagen shield presoaked in 0.5% amphotericin B for one hour. The rabbits were killed after one, three, or five days of treatment. Quantitation of fungi in the cornea was achieved by culturing homogenates and counting colony-forming units. Treatment with amphotericin B applied either as hourly instilled drops or absorbed in collagen shields significantly (P less than .05) reduced corneal fungal counts at all time points when compared to saline-treated control eyes. Rabbit eyes treated with amphotericin B-soaked collagen shields had significantly lower fungal counts compared with hourly instilled amphotericin B drops at Days 1 (P = .02) and 3 (P = .04), but not at Day 5. The collagen shields were as effective in reducing the number of colony-forming units as were amphotericin B drops at Day 5. These data suggest that collagen shields soaked in amphotericin B could be a useful and convenient treatment device in keratomycosis such as that caused by C. albicans.

External devices have been used to enhance drug delivery. This article reviews the role of collagen shields, iontophoresis, and pumps used to deliver ophthalmic medications. Collagen shields have been used to deliver drugs and promote corneal epithelial healing. Presoaked collagen shields deliver many drugs to the eye as well as or better than traditional methods such as frequent topical therapy or subconjunctival injection. The efficacy of drug delivery by collagen shields was demonstrated in animal models of graft rejection and bacterial keratitis. Iontophoresis uses an electrical current to carry an ionized drug across tissue. Transcorneal iontophoresis delivers high concentrations of a drug to the anterior segment of the eye. Transscleral iontophoresis bypasses the lens-iris diaphragm and produces adequate vitreous levels. Pumps deliver fluid to the eye for extended periods of time via a tube with its distal opening in the conjunctival sac, corneal stroma, anterior chamber, or vitreous cavity. Clinical acceptance of the collagen shield for drug delivery to the anterior segment is better than iontophoresis or pumps, probably because the collagen shield is simpler and more convenient to use.

Corneal and conjunctival biopsies of 13 patients with rheumatoid arthritis and corneal ulceration (RA-keratomalacia) have been characterized by immunohistological and histochemical analysis. Biopsies from 13 patients with bacterial conjunctivitis, 7 patients with allergic conjunctivitis, 15 patients with senile cataract and 15 patients with keratokonus served as controls. The phenotypic composition of the conjunctival inflammatory infiltration of rheumatoid corneal ulceration was not significantly different from the other inflammatory eye diseases studied. However, conjunctival epithelial cells of all RA-patients showed strong de novo expression of HLA-DR- and DP-antigens. HLA-DQ-antigens were only weakly expressed in a minority of patients. In bacterial conjunctivitis a less intense HLA-class-II-expression was found to be restricted to HLA-DR-antigens. Furthermore, in RA patients stromal fibroblasts of the cornea expressed lysosomal elastase. Both observations could be explained by paracrine action of interleukins produced by infiltrating T-lymphocytes and macrophages. Thus, it might be tempting to speculate that immunologically induced, elastase mediated autodegradation of corneal stroma may be an important factor in the pathogenesis of rheumatoid corneal ulceration.

In animal studies, bromocriptine (Pravidel) has demonstrated immunosuppression because of inhibition of prolactin. After publication of positive results in the treatment of uveitis in a few case reports and in an uncontrolled study we conducted a double-blind placebo-controlled study in 13 patients with chronic recurrent anterior uveitis who had experienced three or more recurrences during the previous year. Therapy was started gradually during the symptom-free interval and continued for 1 year with 2.5 mg twice daily. Two recurrences or relevant side effects led to discontinuation of the therapy. In the bromocriptine group, 2 of 7 patients had no recurrences during the study period. Two patients had to stop because of two recurrences but, in contrast to earlier recurrences, they responded to local corticosteriod treatment within a few days. The remaining 3 patients (1 with one recurrence, 2 without) had to stop because of the side effects (arterial hypotension, arthritic complaints). In 5 of 6 patients in the placebo group treatment was stopped because of recurrences and in 1 patients because she believed she was experiencing a side effect from bromocriptine (breast atrophy). In summary, bromocriptine seems to have a prophylactic effect on anterior uveitis. Side effects were frequent but mild compared to other immunosuppressives. In case of arterial hypotension, additional therapy could make this drug more tolerable.

Evidence is accumulating that prolactin (PRL) may play a physiological role in the regulation of humoral and cell-mediated immune responses. On the basis of these observations, we measured levels in the serum and aqueous humor of 28 patients with cataract or anterior uveitis with concomitant cataract. Intraocular concentrations were measured in a range from 0.1 to 3.4 ng/ml. Whereas serum PRL levels failed to show significant differences between the two groups (P = 0.39), intraocular concentrations were significantly higher in uveitis patients (P less than 0.001). The level in aqueous humor did not reflect the serum concentration in either group. To the best of our knowledge, this is the first study measuring PRL concentrations in human aqueous humor.

The results of local application of monoclonal antibodies (mAb) in rabbit eyes are presented. To improve intraocular access of the high-molecular-weight protein it was entrapped in large (0.2 microns) unilamellar, negatively charged liposomes. Concentrations of the free or encapsulated drug were measured by ELISA in different eye compartments following repetitive drop administration or single subconjunctival injection. Although mAb became measurable in specimens of conjunctiva and cornea, it was not detectable (less than 0.5 ng/ml) in aqueous humor, lens or the vitreous body. In contrast, concentrations of the liposome-encapsulated drug were measurable as little as 30 min after topical application in the aqueous humor.

A 24-year-old male patient who had been received Fansidar developed exudative erythema multiforme major with massive involvement of the skin, mucous membranes, and both eyes. The chronic inflammation led to proliferation of the conjunctiva, with keratinization of the ocular surface and corneal vascularization. The final result was comparable to cicatricial pemphigoid, with blindness of both eyes. In histological sections, massive lympho-plasma-cellular infiltration with circumscribed perivasculitis was seen. The lymphocyte transformation test was still positive 18 months after onset of the disease. Class II antigens were evident in some of the conjunctival biopsies.

A 29-year-old man with chronic myeloid leukemia and two successfully treated blast crises exhibited ocular symptoms. Eighteen months after the diagnosis of leukemia he presented with unilateral hyperplastic iris stroma, anterior chamber inflammation and vitreous infiltration. Uveitis was diagnosed tentatively, and local therapy was begun with corticosteroids. Because the findings remained unchanged an invasive diagnostic evaluation followed. Although iris biopsy revealed only unspecific mononuclear cell infiltration (mainly T-lymphocytes), selectively aspirated vitreous material contained myeloid cells of different stages of maturation and blast cells. Peripheral blood, liquor and bone marrow showed no signs of acceleration or transition of the primary disease. Thus, an extramedullary blast crises with isolated vitreous infiltration was diagnosed.

The incidence of allograft rejection was determined for 740 penetrating keratoplasties performed between 1980 and 1987. All 740 cases were followed up for at least 1 year. The reaction forms of allograft rejection were grouped according to biomicroscopic appearance. The incidence and progression of symptoms are described. Rejection types are subdivided into patients with favorable versus poor prognosis. Of the patients 37.9% demonstrated an immune response (including discrete forms). Clear reaction patterns within the two groups of patients became apparent when reaction variations were carefully differentiated. Epithelial immune reaction was found in 5.2%/10.5% and subepithelial infiltration in 1.7%/4.8% of the patients with favorable/poor prognosis. The largest disparity in frequency occurred in progressive endothelial reaction; 3.8% in patients with favorable versus 36.7% in patients with poor prognosis. Focal endothelial reactions occurred in both groups with comparable frequencies (14.1%/13.3%). The large percentage of immunological reactions, including late manifestations (approximately 12% after 1 year) and some with irreversible progression, warrants continuing efforts to treat and prevent this complication.

Cyclosporin A eye drops were used on six patients to treat corneal ulcers associated with rheumatic diseases, oculomucocutaneous syndrome, and Sjögren's syndrome. Conjunctival excision was additionally carried out in two cases. All ulcers healed rapidly. The mechanism of ulcer formation involving T-lymphocytes is discussed.

Under certain pathologic circumstances autoantigens lead to the formation of autoantibodies. In uveitis, autoimmunologic phenomena will also be discussed. We studied 149 sera of uveitis patients for various organ specific and non-organ specific autoantibodies. We found antisarcolemal autoantibodies (ASA) mainly in patients with acute iritis (59% positive) but also in panuveitis (40%) and iridocyclitis (29%). On the other hand antiendothelial antibodies (AEA) could be found in 43% of patients with chorioretinitis. Antisinusoidal antibodies (SA) have been detected in iritis and panuveitis patients more often than in the control group which show positive autoantibodies in 5% of all cases. Using ELISA we looked for antibodies against keratin, laminin and microsomes. Because antimicrosomal-, antilaminin- and antisarcolemal-antibodies recognize the same epitope, there was a good correlation of these three autoantibodies. Similar clusters of autoantibodies (ASA, AEA and SA) have been found in various infectious diseases and in chronic inflammatory diseases in which an infectious component is discussed. These results may indicate that iritis, iridocyclitis, chorioretinitis and panuveitis are secondary reactions of eye tissue following a systemic primary disease. Especially viruses are well-known for their production of autoantibodies. In patients with intermediate uveitis we could not demonstrate these autoantibodies more often than in the control group, favouring theories which believe in an autoimmune reaction against vitreous elements.

Chronic recurrent uveitis still responds unsatisfactorily to therapy. Prolactin, a pituitary hormone that attains high blood levels physiologically in stress situations, has an immune modulating effect. The prolactin antagonist bromocriptine (2 x 2.5 mg/day) was administered prophylactically to prevent recurrence in seven patients with chronic iritis/iridocyclitis or panuveitis (Group A). On this therapy two patients suffered a mild recurrence (in one case in the first month). Another patient developed rebound uveitis after discontinuing bromocriptine medication. Tolerance appears to vary considerably, and in three patients the therapy had to be discontinued because of arterial hypotension and congestive rhinopathy, respectively. The three patients in Group B (with iritis, sympathetic ophthalmia, and intermediate uveitis) received bromocriptine in addition to cyclosporin A after plasmapheresis. This combination resulted in an elevated cyclosporin A plasma level, and the dosage was reduced by 30-50% (plasma level 70-120 ng/ml). With this lower dosage of cyclosporin A, the creatinine level of one of the patients returned to normal. Viewed on the basis of these initial results bromocriptine would appear to reduce the number of recurrences of chronic uveitis. Moreover, the synergism with cyclosporin A, described both theoretically and in an animal model in the literature, appears attainable in therapy.