Comparison of a non-preserved 0.1% T-Gel eye gel (single dose unit) with a preserved 0.1% T-Gel eye gel (multidose) in ocular hypertension and glaucomatous patients.

AIM: This comparative, open design, phase III study was to assess the non-inferiority of the non-preserved T-Gel 0.1% single dose unit (SDU) versus its preserved multidose (MD) reference. METHODS: 175 patients with bilateral POAG or OHT were randomised: 87 patients were to receive one drop daily of T-Gel 0.1% MD and 88 patients were to receive one drop daily of T-Gel 0.1% SDU, for a treatment period of 12 weeks. The primary efficacy variable was the change in intraocular pressure (IOP) in the worse eye between the baseline and the last assessment. Subjective and objective ocular signs as well as adverse events were recorded for safety. Global tolerance was assessed by the investigator and by the patient. RESULTS: The mean percentage reduction from baseline IOP was 24% for both treatments groups, which was consistent with previous studies. The safety results were comparable in both treatment groups. Because of gel formulation, mild short lasting episodes of blurred vision occurred for about 20% of patients. The global tolerance assessment reported that both treatments were well tolerated. CONCLUSION: The overall study results demonstrated that T-Gel 0.1% SDU is not inferior to T-Gel 0.1% MD.

Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.

We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.

Absence of tolerance to noninherited maternal transplantation antigens expressed on skin and cornea.

Tolerance to noninherited maternal antigens (NIMAs) of the MHC was investigated in a rat model involving both skin and corneal transplants. Recipient animals were obtained by backcrossing F1 hybrids to parental strain animals. In one group of experiments, crosses were (DA[RT1a] x LEW[RT1(1)]) female-to-LEW male and, in a second group, (DAxPVG[RT1c]) female-to-PVG male. Homozygous backcross offspring (RT1(1/1) or RT1c/c) were putatively tolerant to DA NIMAs if the mother was a hybrid animal, having been exposed to these antigens in utero. The equivalent offspring of hybrid fathers, i.e., LEW female x (DAxLEW) male or PVG female x (DAxPVG) male, served as putatively nontolerant controls. Hemagglutinating antibody levels were measured against the class I RT1Aa antigen on days 7 and 14 after up to three consecutive subcutaneous DA strain skin grafts. Significantly lower titers were found in the putatively tolerant group 7 days after the first skin graft in the RT1a-to-RT1(1) combination (P < 0.05). Levels were not significantly different at any other time point, or at any time point in the RT1a-to-RT1c combination. Tolerance to a corneal graft was not demonstrated in either the strongly rejecting RT1a-to-RT1(1) combination or weakly rejecting RT1a-to-RT1c, whether or not animals were presensitized to RT1a antigens with DA skin grafts. We conclude that tolerance to NIMAs is unimportant in this clinical rat model of transplantation.

Influence of donor and histocompatibility factors on corneal graft outcome.

The Corneal Transplant Follow-up Study has followed 2311 penetrating keratoplasties for up to 450 days after transplant. A total of 207 failures were observed, including 65 classical rejections and 35 endothelial decompensations. At 12 months, graft survival was 89%, and survival free from rejection was 87%. For surviving grafts, risk of failure reduced from 4.8% in the first 75 days and stabilized after 5 months at 1.2% in each 75-day interval. Risk of rejection initially followed a similar pattern, but then increased after 12 months. Multifactorial analyses accounted for differences in recipient characteristics and interrelationships of donor factors. Donor age, sex, cause of death, and method of corneal storage were not found to influence significantly either time to graft failure or time to first rejection. Grafts in prospectively tissue-typed donor-recipient pairs were generally considered before surgery to be at increased risk of either graft failure or rejection. With due allowance, increasing risk of rejection was associated with increasing numbers of mismatches at HLA-A and HLA-B broad antigens. The opposite was true at HLA-DR broad antigens, where increased risk of rejection was observed with no mismatches.

Immune cell infiltration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes simplex virus type 1.

Following inoculation of the mouse cornea with herpes simplex virus type 1 (HSV-1), the spread of virus was investigated and the types of immune cell infiltrating the trigeminal ganglion (TG) were identified in low temperature paraffin wax sections. Virus antigen was first found on day 3 and was absent after day 14. Early presentation of antigen to T cells may occur since increased expression of major histocompatibility complex (MHC) class II antigens, including de novo expression on satellite and Schwann cells, was detected in foci of such antigen on day 3. A second large peak of such expression was detected on day 10 together with increasing numbers of B and T cells. Large numbers of these lymphocytes and extensive expression of MHC class II were seen in the TG well into the phase of virus latency; the significance of this is discussed.

Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.

We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.

Factors influencing the suitability of organ-cultured corneas for transplantation.

PURPOSE: To assess the influence of donor and storage factors on the suitability of organ-cultured corneas for penetrating keratoplasty (PKP) using multifactorial regression analysis. METHODS: Corneas (mean donor age, 57 years; standard deviation, 21 years) were stored by organ culture at 34 degrees C for up to 5 weeks (mean, 22 days; standard deviation, 6 days). The endothelium was assessed by light microscopy, and corneas with < 2200 cells/mm2 were considered unsuitable for PKP. RESULTS: Of the 9250 corneas stored between 1992 and 1994, 59% were issued for PKP, 5% were discarded because of bacterial or fungal contamination, and 30% were unsuitable for PKP owing to endothelial deficiencies. Donor age had the strongest influence on suitability for PKP: > 80% of corneas from donors younger than 40 years of age were issued for PKP compared with only 45% of corneas from donors 80 years of age and older. There was an overall decline in the percentage of corneas suitable for PKP with increasing storage time, but the rate of this decline was inversely related to donor age. Cause of death and post mortem times to enucleation and to storage had only a small influence on suitability for PKP. CONCLUSIONS: Criteria based on endothelial assessment rather than on donor age allow corneas from donors of all ages, stored by organ culture for extended periods, to be used for PKP. Organ culture also allows corneas with bacterial or fungal contamination to be identified and discarded before they are grafted.

Effect of mismatches for major histocompatibility complex and minor antigens on corneal graft rejection.

The importance of minor histocompatibility genes in corneal graft rejection was investigated using a model that simulates the major histocompatibility complex (MHC) and minor mismatches of the human allograft more accurately than previous animal models. DA(RT1a) x LEW(RT1(1]F1 hybrid rats were backcrossed to LEW, and the backcross generation were used as corneal graft recipients. Female DA(RT1a) strain animals were used as donors throughout. As in humans, the MHC disparity (a to 1) between each donor-recipient pair could be controlled; minor mismatches were variable and unknown. The MHC haplotype of each backcross individual (either homozygous l/l) or heterozygous a/l) was determined. Depending on this haplotype, the transplanted DA cornea was either matched or mismatched with the recipient for MHC antigens. The average proportion of minor disparate loci was 50%, although this was variable and unknown from recipient to recipient. Some animals of each MHC type were sensitized with three subcutaneous DA strain skin grafts at intervals of 2 weeks. Prior sensitization caused more rapid corneal graft rejection in both MHC mismatched (P less than 0.001) and matched (P less than 0.01) animals. All animals in the two MHC-mismatched groups (sensitized, 26; unsensitized, 17) and most in the MHC-matched groups (sensitized, 25 of 27; unsensitized, all 13) rejected their grafts. The MHC matching resulted in a greater range of survival times, although the difference in survival in unsensitized animals between matched and mismatched groups was not significant (unsensitized, P greater than 0.05; sensitized, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine production in a murine model of recurrent herpetic stromal keratitis.

PURPOSE: To determine the pattern of cytokine production in the cornea and its relationship with viral antigens, in our murine model of recurrent ocular herpes simplex virus (HSV)-1 infection. METHODS: Six weeks after corneal inoculation with HSV-1, the eyes of latently infected and control mice were UV irradiated and examined for signs of disease and viral reactivation. The eyes of five mice with recurrent stromal disease and two controls were processed for immunohistochemistry on days 4, 7, 10, and 14 after irradiation. Sections were double stained for viral antigens and one of the following cytokines: interleukin (IL)-1ss, IL-2, IL-4, IL-6, IL-10, IL-12, and interferon (IFN)-gamma. RESULTS: Fifty percent of mice showed signs of recurrent stromal disease, the severity of which peaked on day 10 after UV irradiation. There was a large cellular infiltrate in the stroma of all the corneas with recurrent disease and the predominant cytokines were IL-1ss, IL-6, IL-10, IL-12, and IFN-gamma, all present in large numbers of cells on the days studied. There were very few cells producing IL-2 and IL-4. Control eyes had no significant cytokine-producing cells in the stroma. CONCLUSIONS: These observations suggest that recurrent herpetic stromal keratitis (HSK) may not be characterized by a classic T-helper (Th)1 or Th2 response. However, the large number of IFN-gamma(+) and IL-12(+) cells and the relative absence of IL-4 favors a Th1 response, and despite the numerous IL-10(+) cells, the overall balance of cytokine production appears to be proinflammatory.

Recurrent herpes simplex after corneal transplantation in rats.

PURPOSE: To ascertain the effect of trauma from surgery and rejection on the incidence and timing of recurrent herpes simplex virus (HSV) disease after corneal transplantation. To locate virus antigen and identify cells of the immune system infiltrating corneas with recurrent disease. METHODS: PVG rats were inoculated on the cornea with HSV-1 McKrae. Recurrent disease was induced either by ultraviolet (UV)-irradiation of the cornea or by corneal transplantation. After corneal transplantation, animals shedding virus in the tear film were killed on days 1 to 4 of shedding. Eyes were fixed, embedded, sectioned, and stained for virus antigens, infiltrating cells, major histocompatibility complex class II, and adhesion molecule molecule expression. RESULTS: In the first 15 days after corneal transplantation, 8 of 91 rats shed virus, and between days 16 and 30, an additional 3 of 60 rats shed virus (12% of total rats, comparable to the percent that shed after UV irradiation). Shedding sometimes was accompanied by punctate epithelial lesions in the recipient cornea and stromal opacity. The rejection process itself did not induce or exacerbate recurrent disease. In all corneas examined from eyes that shed virus, antigen was found in several locations at the graft-host junction, sometimes in the absence of clinical signs of disease, and frequently it extended through the stroma to the endothelium. Granulocytes were the main infiltrating cell in areas of virus antigen. CONCLUSIONS: Corneal transplantation trauma is a stimulus to recurrent disease of similar potency to UV irradiation. The graft-host junction is an area in which virus spreads easily and can reach the endothelium readily. In humans, the incidence of recurrent disease at this location may be greater than has been recognized.

Corneal sensitivity and substance P in experimental herpes simplex keratitis in mice.

Experimental herpes simplex keratitis in the mouse produced a rapid fall in both corneal sensitivity and levels of corneal substance P (SP). This finding supports the association of SP with sensory neurones and shows that such levels can be used as an indication of damage to neurones resulting, for example, from infection with HSV. However, the delay in recovery of SP compared to the more rapid and complete recovery of sensitivity suggests that SP in the cornea is not directly involved in mediation of the blink reflex.

Ocular infection with herpes simplex virus in several strains of rat.

PURPOSE: To assess the suitability of the rat for studies of ocular infection with herpes simplex virus (HSV). METHODS: LEW, AO, DA, PVG, and (DAxLEW)F1 x LEW backcross generation rats, 7 to 9 weeks of age, were inoculated with HSV-1 McKrae. The course of primary disease was assessed by clinical observation using a slit lamp. Infectious virus was assayed in ocular and nervous tissue, and the incidence of latent infection was determined. RESULTS: LEW and AO strains were the most susceptible. All LEW rats died after an inoculum of 4 x 10(2) plaque-forming units (pfu) and developed severe corneal disease and uveitis. In contrast, all PVG rats survived 10(4) pfu, 60% survived 4 x 10(4) pfu, and eye disease was restricted to epithelial lesions, sometimes accompanied by mild stromal haze. This resolved, even in animals that developed central nervous system disease. The DA strain showed intermediate susceptibility. Resistance was dominant because disease in backcross generation (DA x LEW)F1 x LEW rats resembled that of the DA rather than the LEW strain. Resistance appeared to be linked to coat color (P < 0.001) rather than to major histocompatibility complex (MHC) type. Chronic stromal disease did not occur in survivors (DA, PVG, and hybrid strains only). CONCLUSIONS: The susceptibility of rat strains to infection of the cornea with HSV varies, and, as with mice, resistance seems to be controlled by non-MHC genes. Rats may prove useful for immunologic studies. Virus reactivation will be the subject of a future report.

Immunological aspects of corneal graft rejection.

Immunological graft rejection is a major cause of corneal graft failure. HLA class I and II antigens are expressed by various cells within the cornea and during sensitisation of the recipient donor antigens appear to be presented by both donor and recipient antigen presenting cells. Certain donor and host factors have been shown to influence the incidence of corneal graft rejection, and the manipulation of these factors is discussed.

Ocular infection with herpes simplex virus in nonimmune and immune mice.

In a detailed study of ocular infection by herpes simplex virus (HSV) type 1 in mice, the course and signs of eye disease were investigated and compared in primary and secondary infection using slit-lamp examination, culture of the tear film, and monitoring of the blink reflex. Response to primary inoculation ranged from subclinical infection to severe keratitis. Compared with conjunctival scarification, corneal scarification resulted in more frequent and severe eye disease and signs of CNS infection. Previous infection in the skin of the contralateral ear considerably modified subsequent infection of the eye so that signs of disease occurred earlier, were limited to dendritic keratitis with some stromal involvement, and were largely reversible. The mouse seems to be a suitable animal for studying ocular infection with HSV.

Antigens of herpes simplex virus in whole corneal epithelial sheets from mice.

Mice were inoculated with herpes simplex virus in the skin of the snout or by scarification on the cornea and then examined for eye disease using a slit lamp. Whole mounts of corneal epithelium were stained for virus antigens by the peroxidase-antiperoxidase method, and infectious virus was isolated from eyewashings. Antigens were present one day after corneal inoculation, but after inoculation of the snout, there was a delay of three days before antigens were seen. This delay and the distribution of antigens were evidence of zosteriform spread from the snout to the eye via the nervous system. Disease of the cornea varied in severity and timing depending on the site of inoculation. The peroxidase-antiperoxidase method was more sensitive than isolation of virus from eyewashings and allowed the site and distribution of infected cells to be seen.

Severe persistent inclusion conjunctivitis in a young child.

A 14-month-old girl had inclusion conjunctivitis although there had been no signs or symptoms that required medical attention in her first year. By 2 1/2 years of age, the child had extensive pannus and corneal scarring that severely reduced vision. The infection was caused by a genital strain of Chlamydia trachomatis, TRIC type E, that was probably acquired at birth. Although genitally transmitted chlamydial strains normally cause a self-limiting inclusion conjunctivitis in areas where trachoma is not endemic, this case illustrates that they may occasionally cause severe trachoma. Possibly local idoxuridine (IDU) treatment (administered to this patient before the correct diagnosis was made) contributed to the severity of this infection.

Clinical evaluation of twice-daily emedastine 0.05% eye drops (Emadine eye drops) versus levocabastine 0.05% eye drops in patients with allergic conjunctivitis.

PURPOSE: The efficacy and safety of emedastine 0.05% eye drops (Emadine; Alcon Laboratories, Inc, Fort Worth, Texas), a new H(1) antagonist, were studied in comparison to levocabastine 0.05% eye drops (Livostin; Janssen-Cilag N V, Berchem, Belgium) during a twice-daily treatment schedule for 6 weeks in adult and pediatric patients with seasonal allergic conjunctivitis. METHODS: In a prospective, multicenter, randomized, double-masked, parallel group study, 222 patients with allergic conjunctivitis were randomized (221 received treatment) to either emedastine or levocabastine, instilled twice daily for 6 weeks. Patient diaries were completed four times daily (before the morning and evening instillations, at noon, and in the afternoon), and clinical examinations were conducted at regular intervals. Primary efficacy variables of ocular redness and itching and secondary efficacy variables of chemosis, eyelid swelling, patient diary data, and physician's global assessment were analyzed. RESULTS: Both emedastine and levocabastine produced a statistically significant (P =.0001) reduction in itching and redness within 5 minutes of the first instillation. All signs and symptoms improved progressively over the 6-week treatment period. After 7 days of use, and throughout the remainder of the study, emedastine was statistically superior to levocabastine (P <.006) in preventing and alleviating the signs and symptoms (itching, redness, chemosis, and eyelid swelling) of allergic conjunctivitis. CONCLUSIONS: Emedastine 0.05% eye drops administered twice daily are more efficacious than levocabastine 0.05% eye drops in the prevention and treatment of the signs and symptoms of allergic conjunctivitis in adults and children of 4 years and above. Both emedastine 0.05% eye drops and levocabastine 0.05% eye drops were well tolerated.

Experimental Acanthamoeba keratitis: II. Immunohistochemical evaluation.

In a Wistar rat experimental model of Acanthamoeba keratitis immunohistochemical techniques were used to analyse the host cellular response. The inflammatory cell profile was observed to change at intervals. In tissue sections the cellular response consisted of neutrophils on the first day but predominantly macrophages on the following days. Some T lymphocytes but no B lymphocytes were observed.

Experimental ulcerative herpetic keratitis. I. Systemic immune responses and resistance to corneal infection.

The influence of previous infection with herpes simplex virus (HSV) on the susceptibility of rabbits to corneal inoculation of the virus was studied by means of the microtitration model for ulcerative herpetic keratitis. Systemic immune responses were assayed after skin and eye infections by means of the lymphocyte transformation (LT) and complement fixation (CF) tests. Both cutaneous and ocular primary infections resulted in cellular and humoral immune responses to HSV. In comparison to primary ocular infection, corneal disease in rabbits with previous skin infection ('secondary corneal infection') resulted in earlier initiation of cellular and humoral immune responses, while complement-fixing antibody titres reached a higher level. A previous cutaneous infection ('immunisation') provided considerable protection to the cornea and also accelerated recovery from corneal ulcerative disease. A correlation was observed between the initiation of the lymphocyte transformation response and the beginning of healing of corneal ulceration. A previous unilateral ocular infection induced an even higher degree of corneal resistance in that eye, and the opposite eye was protected to the same extent as by cutaneous immunisation.

Experimental ulcerative herpetic keratitis. III. Evaluation of hyperimmune gammaglobulin therapy.

The value of hyperimmune gammaglobulin (HGG) therapy in ulcerative herpetic keratitis was assessed in rabbits. HGG treated of early disease in normal rabbits was very effective, producing a 10-fold rise in the virus concentration needed to infect 50% of sites (CID50) and an 88% inhibition of ulceration after 2 days. The efficacy of the gammaglobulin preparations tested depended on their anti-HSV antibody content. Established disease was considerably less responsive to HGG therapy. No conclusive effect of HGG therapy could be demonstrated in rabbits with a previous HSV skin infection ('immunised'). Corticosteroid-induced geographic ulceration in immunised rabbits was not prevented by concurrent HGG therapy. The findings indicate that HGG is unlikely to represent a useful therapy for ulcerative herpetic keratitis but that it may be valuable in primary disease and in long-term prophylaxis.