The effect of air tamponade on the ingress of ocular surface pathogens in sutureless transconjunctival microincisional vitrectomy.

PURPOSE: To assess the effect of air versus fluid tamponade on the intraocular migration of india ink particles through nonsutured transconjunctival 23-gauge sclerotomies in living porcine eyes. METHODS: Both eyes (n = 20) underwent 3-port, transconjunctival, 23-gauge vitrectomy. An angled trocar insertion technique was used in all cases. In each animal, one eye underwent a partial fluid-air exchange at the conclusion of the vitrectomy, yielding an air fill of approximately 80% (n = 10), whereas the other was left fluid filled (n = 10). After removal of the instruments and trocar cannulae, india ink was applied to the ocular surface. Animals were allowed to partially recover from anesthesia and resume normal blinking behavior. Animals were then reanesthetized, euthanized, and enucleated. Histopathologic examination was performed in a masked fashion. The presence and location of ink was noted for each identified sclerotomy. RESULTS: Ink was identified on the ocular surface in 18 of 20 eyes. Sclerotomy wounds were identified in 16 of 20 eyes. Ink penetration was seen in 2 of 16 sclerotomy wounds, 1 in an air-filled globe and 1 in a fluid-filled globe. In both eyes, the ink was identified along the outer one third of the wound. There was no penetration of ink along the inner two thirds of the sclerotomy wound or in the posterior segment of any eyes. CONCLUSION: In an experimental, in vivo, porcine model, india ink migration into angled transconjunctival sclerotomy incisions was minimal, regardless of the use of an 80% fluid-air exchange at the conclusion of the case.

Spontaneous induction of immunoregulation by an endogenous retinal antigen.

PURPOSE: Ocular immune deviation studies have relied on intraocular injections of antigen to induce altered immune responses. Contributions of the injection process itself have complicated study of the mechanisms and interpretations of biological significance. In the current study, transgenic mice were used to determine the presence of immune deviation or other evidence of immunoregulation, elicited by endogenous Escherichia coli beta-galactosidase (beta-gal) expressed through a retinal arrestin promoter. METHODS: Mice that express beta-gal in the retina and various control mice were immunized with beta-gal and tested for immune responsiveness by the ear-swelling test for delayed-type hypersensitivity (DTH), in vitro proliferation assays, and cytokine assays. Spleen cells from transgenic mice were also transferred to normal recipients to test for transfer of immune deviation. RESULTS: Endogenous retinal beta-gal expression depressed the DTH response and proliferation assays after beta-gal immunization. The ability to depress DTH was transferred by naïve spleen cells from transgenic mice to nontransgenic mice. Use of an immunization protocol that included high-dose Mycobacterium tuberculosis (M tb) H37Ra adjuvant and concurrent administration of pertussis toxin reversed the inhibition of DTH. CONCLUSIONS: An endogenous neo self-retinal antigen alters subsequent immune responses without intraocular injection, suggesting that normal retinal proteins in quiet eyes induce immunoregulatory responses. Based on cytokine assays, there were similarities to the immune deviation induced by intraocular inoculation in the IL-4 response, but the IL-10, IFN-gamma, and TGF-beta1 results were not similar, indicating that the mechanisms differ. The ability of supplemented adjuvant to overcome endogenous tolerance may be related to the requirement for supplemented adjuvants in the induction of experimental autoimmune uveoretinitis.

Disposition of WR-1065 in the liver of tumor-bearing rats following regional vs systemic administration of amifostine.

PURPOSE: Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. It was hypothesized that amifostine may be a good candidate for regional drug delivery to the liver because of its large hepatic extraction and total body clearance. METHODS: Rat livers were implanted with Walker-256 tumors. The tumor-bearing rats received 15 min infusions of amifostine (200 mg/kg) via the portal vein or the femoral vein. WR-1065 concentrations in the blood, liver and tumor were measured at various times. RESULTS: The WR-1065 tumor portal dosing AUC15-60 was 40% of systemic dosing, and tumor concentrations following portal dosing were one-fifth of that following systemic dosing. The portal dosing WR-1065 liver AUC15-60 was 60% higher than the values for systemic dosing. The liver/tumor concentration ratios of WR-1065 following portal dosing were up to 8-fold higher than the ratio following systemic administration. Unfortunately, systemic exposure to WR-1065 was greater following portal vs systemic amifostine. CONCLUSIONS: Amifostine may provide increased liver protection and decreased tumor protection from radio- or chemotherapy when administered by the portal vein. However, portal dosing also increases systemic exposure to WR-1065, which is associated with hypotension.

Resting CD8 T cells recognize beta-galactosidase expressed in the immune-privileged retina and mediate autoimmune disease when activated.

Although the expression of class II major histocompatibility complex (MHC) in retina is extremely low, it is an established fact that activated CD4 T cells, specific for retinal antigens (Ags), mediate experimental autoimmune uveoretinitis (EAU). Conversely, CD8 T cells have not been shown to recognize Ag in the retina. This study investigated whether retinal-specific Ags are detected by class I MHC-restricted CD8 T cells. Using a CD8 T-cell clone (beta3) specific for an immunodominant epitope of beta-galactosidase (beta-gal), local Ag recognition was shown by transfer of activated beta3 cells into beta-gal transgenic (Tg) mice expressing beta-gal in the retina (hi-arr-beta-gal mice), or in the brain and eye (GFAP-beta-gal mice). Beta-gal-positive photoreceptor cells were damaged in the retina of hi-arr-beta-gal mice, and anterior segment disease was found in the eyes of GFAP-beta-gal mice. Ag recognition by resting CD8 T cells was also evaluated. Recovery of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labelled beta3 cells from hi-arr-beta-gal mice was slightly decreased compared to recovery from B10.A mice, while recovery from GFAP-beta-gal mice was transiently increased. Conversely, recovery of CFSE- cells increased in hi-arr-beta-gal mice, consistent with an Ag-dependent response. The CFSE content of the CFSE+ population was unchanged relative to beta3 cells recovered from controls. Intracellular cytokine responses of beta3 cells recovered from hi-arr-beta-gal and GFAP-beta-gal mice correlated with the number of cells recovered, regardless of CFSE content. Even though their production of interferon-gamma and tumour necrosis factor-alpha was affected little by transfer into hi-arr-beta-gal recipients, the ability of beta3 cells to mediate delayed-type hypersensitivity was inhibited in hi-arr-beta-gal mice. These results show that resting CD8 T cells are affected by the presence of Ag that originates in retina and, when activated prior to transfer, mediate pathogenic autoimmunity against retinal and other ocular targets.

Regional pharmacokinetics of amifostine in anesthetized dogs: role of the liver, gastrointestinal tract, lungs, and kidneys.

Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. In this study, we characterized the sites and extent of organ-specific activation by the liver, gastrointestinal tract, lungs, and kidneys after systemic administrations of amifostine. A total of 10 dogs were infused via the cephalic vein using sequential dose rates of drug at 0.125, 0.500, and 1.00 micro mol/min/kg. Infusion of each dose rate lasted 2 h, at which time steady-state plasma concentrations were obtained (i.e., portal vein, carotid artery, hepatic vein, pulmonary artery, and renal vein). The hepatic arterial, portal venous, and renal arterial blood flows, and cardiac output, were measured. The hepatic and splanchnic extraction of amifostine remained high at 90%, whereas gastrointestinal extraction decreased from 43 to 12 to 15% with increasing dose. Pulmonary extraction of amifostine was low at 7%, whereas renal extraction was intermediate at 57%. Because blood flow measurements were relatively constant during the drug infusions, clearance parameters paralleled that of organ extraction. As a result, saturability was observed in the gastrointestinal blood clearance (i.e., from 9.8 to 2.8-3.3 ml/min/kg) and total body plasma clearance of amifostine (i.e., from 52.6 to about 37.3 ml/min/kg), as the doses increased. Due to the drug's high activation in liver, these findings suggest that amifostine may offer good protection of this organ against the toxicities of chemotherapy and radiation.