Nature of lymphocyte-tumor interaction. A general method for cellular immunoabsorption.

The binding of sensitized lymphocytes to tumor cells that leads to tumor cell lysis in vitro has been investigated using poly-L-lysine-fixed tumor cell monolayers and lymphocytes obtained from the anatomical site of tumor allograft rejection. The results show that magnesium is an important prerequisite for this interaction and that the extent of lymphocyte-tumor cell binding depends upon temperature as well as pH. Binding can occur in the absence of serum, although serum factors are necessary for the completion of the cytolytic process. The poly-L-lysine technique is applicable to the formation of confluent monolayers with virtually any normal or neoplastic cell type, including those that are otherwise nonadherent to surfaces. Cells immobilized by this technique can be used for the specific immunoabsorption and subsequent recovery of effector lymphocytes sensitized against transplantation or tumor cell antigens.

A computerized system for the selection of organ transplant recipients.

The Southeastern Regional Organ Procurement Program has developed a computerized system for the selection of organ transplant recipients. This system has proven to be a useful method for the rapid selection of histocompatible recipients for organ transplants from a large pool of potential recipients in a wide geographical area. In addition, the system provides a readily accessible source of data that can be used to analyze the relationship between histocompatibility and transplant survival.

Recovery of herpes simplex virus from ocular tissues of latently infected inbred mice.

Evidence for latent infection of ocular tissues following topical corneal inoculation with herpes simplex virus type 1 (HSV) was sought in three strains of inbred mice that differ in susceptibility to HSV stromal keratitis. Corneas of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV. At 6-8 weeks after inoculation, when no active ocular infection was present, minced whole eyes and trigeminal ganglia were assayed for latent virus. Virus was recovered by explantation from minced eyes of all three strains (DBA/2 = 20%; BALB/c = 17%; C57BL/6 = 7%). In order to determine which ocular structures harbored virus, corneas, retinas and choroid-sclera were cultivated separately. Virus was activated from corneas of DBA/2 and BALB/c mice, but not from corneas of C57BL/6 mice. These findings suggest that HSV is capable of establishing latent infection in ocular tissue of inbred mice and that the rate of establishment of latency is under host genetic control. Since neural cell bodies are not present in the cornea, the data suggest that latency is established in cells other than neurons.

Corneal neovascularization induced by stimulated lymphocytes in inbred mice.

The intrastromal implantation of Concanavalin A-stimulated allogeneic lymphocytes induced corneal neovascularization (CNV) in inbred C57BL/6 and BALB/c mice. Control syngeneic stimulated, allogeneic non-stimulated, and allogeneic stimulated irradiated lymphocytes were not angiogenic. CNV induced by allogeneic stimulated lymphocytes in BALB/c recipients was significantly greater than in C57BL/6 recipients. This response reflected host-versus-graft reactivity, since parental recipients responded to F1 hybrid donors, while F1 hybrids did not respond to parental donors. The ability of stimulated lymphocytes to induce CNV may be important in allograft rejection, herpes simplex keratitis, and other corneal immune reactions. The mouse cornea is an excellent model for studying immunologically mediated neovascularization under genetically controlled conditions.

The distribution of HLA antigens on human corneal tissue.

Frozen sections of human corneas, as well as cultured cells derived from the epithelial, stromal, and endothelial layers, were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens using mouse monoclonal antibodies in an indirect immunofluorescence assay. On frozen sections, class I antigens were readily detected on corneal epithelium and keratocytes. Class I antigens were not detected on endothelial cells on frozen sections of adult corneas, but were identified on endothelial cells from some individuals less than 2 years of age. Class II antigens were not detected on corneal epithelial, stromal or endothelial cells on frozen sections. However, HLA-DR-positive dendritic cells were seen in corneal epithelium and were more numerous near the limbus. HLA-DR was expressed by cuboidal cells in the basal layer of conjunctival epithelium from several infants. Cultured cells derived from corneal epithelium, stroma, and endothelium consistently expressed class I but not class II antigens.

Modulation of HLA antigen expression on corneal epithelial and stromal cells.

Human corneal epithelial cells and stromal fibroblasts in culture were incubated with gamma interferon or with medium conditioned by phytohemagglutinin (PHA)-stimulated mononuclear cells. The corneal cells were placed into suspension, assayed for class I (HLA-A,B,C) and class II (HLA-DR) antigens by indirect immunofluorescence, and analyzed with flow cytometry. Epithelial cells treated for 5 days with conditioned medium (CND-M) did not exhibit an increase in class I or an induction of class II antigen expression, although a trend toward increased class I antigen expression was present. Epithelial cells treated for 5 days with 250-500 U/ml of gamma interferon did not demonstrate an increase in class I but did show an induction of class II antigen expression; again, however, a trend toward increased class I antigen expression was present. Stromal fibroblasts treated for 3-5 days with CND-M exhibited an increase in class I antigen expression, but stromal fibroblasts treated for 1-5 days with CND-M did not show an induction of class II antigen expression. Stromal fibroblasts incubated for 1-5 days with 250-750 U/ml of gamma interferon demonstrated both an increase in class I and an induction of class II antigen expression. These data suggest that host lymphokines may intensify the process of corneal graft rejection by augmenting class I antigen expression on allogeneic cells. Moreover, the induction of class II antigen expression by host lymphokines on cells in transplanted corneal tissue may lead to host sensitization and subsequent allograft rejection.

Analysis of human corneal IgG by isoelectric focusing.

Parameters which regulate the localization and retention of IgG within the corneal stroma are complex and poorly understood. Although multiple factors are involved, electrostatic interactions between IgG and anionic corneal tissue components, ie, proteoglycans (PG) and glycosaminoglycans (GAG) may regulate the distribution of antibodies within the corneal stroma. Isoelectric focusing (IEF) and blotting analysis of IgG revealed a restricted pI profile for both central and peripheral regions of the normal cornea. Similar analysis of pathological corneas from keratoplasty specimens in Fuchs' dystrophy and keratoconus reveal a variable IEF profile. In the majority of keratoplasty specimens from patients with corneal edema or graft rejection, there was generally little or no IgG detectable. These results suggest that in edematous corneas where there is altered PG/GAG in the stroma and modified fluid dynamics, there is a concomitant loss of IgG. These findings may have implications for immunologic surveillance and protection of the avascular cornea.

Patterns of herpes simplex keratitis in inbred mice.

The authors have investigated the course of herpes simplex type 1 (HSV) keratitis in three different inbred strains of mice infected with four different HSV isolates. Severity of ocular disease and mortality is dependent upon both the virus isolate and the host strain. In particular, the likelihood of progression from self-limited dendritic keratitis to severe necrotizing stromal keratitis varies markedly among the virus-host strain combinations tested. When mice from strains resistant to stromal disease are crossed with mice from strains susceptible to stromal disease, the F1 offspring are resistant, suggesting that the gene(s) controlling resistance is dominant. Corneal stromal keratocytes and embryo fibroblasts from inbred mice differ significantly in their ability to support the replication of HSV in vitro. HSV replicates more efficiently in vitro in keratocytes from mice susceptible to stromal keratitis than it does in keratocytes from mice resistant to stromal keratitis. These findings provide evidence in an animal model for both virus- and host-related mechanisms that determine susceptibility to stromal keratitis.

The severity of herpes simplex viral keratitis in mice does not reflect the severity of disease in humans.

Four herpes simplex type 1 virus (HSV) isolates were selected from patients with mild ocular disease and four from patients with severe ocular disease on the basis of the number of epithelial recurrences, presence or absence of stromal disease, visual acuity, and the need for corneal transplantation. The scarified right corneas of 20 BALB/c mice were inoculated with each low-passage HSV isolate (1.0 x 10(7) plaque-forming units/ml) and examined three times per week for 2 weeks for the presence and severity of epithelial and stromal disease. The eight individual virus isolates differed with respect to the incidence of dendritic disease (P less than 0.001), the severity of dendritic disease (P less than 0.001), the incidence of stromal disease (P = 0.002), and the severity of stromal disease (P = 0.001) they produced in the mouse. The severity of disease was compared for the two groups of viruses: (1) those that had caused mild disease in their human hosts and (2) those that had caused severe disease. There were no statistically significant differences in the severity or incidence (44 versus 43 animals, respectively) of dendritic disease or stromal disease (27 of 80 animals in each group) between the two groups. These data suggest that the naive BALB/c mouse model of acute HSV keratitis after topical ocular inoculation does not reflect clinically significant differences in the severity of human HSV keratitis that might be caused by variations in the virus genome.

Spread of HSV and establishment of latency after corneal infection in inbred mice.

The spread of herpes simplex virus (HSV) through neural tissues was studied in three inbred mouse strains that differ in susceptibility to HSV stromal keratitis. The left eyes of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV type 1. The optic and trigeminal nerves, trigeminal ganglia, and eyes were assayed for infectious virus on days 1, 2, 3, 4, 7, 9, 11 and 14 after inoculation. At 2-4 months post-inoculation, eyes and trigeminal ganglia were assayed for latent virus. Up to 7 days post-inoculation, infectious virus was present at a similar frequency in the inoculated eyes of mice from all three strains. The quantity of virus recovered, however, was mouse strain-dependent: DBA mice yielded the most virus; C57BL/6, the least. The frequency of virus recovery and the quantity of virus recovered from trigeminal nerves and ganglia also varied according to mouse strain. Infectious virus was recovered from the uninoculated right eye of some DBA and C57BL/6 mice 1 wk after inoculation. The overall incidence of latency differed among inbred mouse strains. However, in mice that developed ocular disease (blepharitis, dendritic keratitis, or stromal keratitis), there was no host strain-related difference in the incidence of latency. These results support the hypothesis that host genetic factors play a role in controlling HSV replication and the spread of virus to neural tissues after ocular HSV inoculation. This control may influence the development and severity of disease. However, once infection occurs, latency is established in both susceptible and resistant mouse strains.

Comparative replication of HSV-1 in BALB/c and C57BL/6 mouse embryo fibroblasts in vitro.

Herpes simplex virus type 1 (HSV)-host cell interactions were studied in fibroblasts from inbred mice by measuring virus replication, virus adsorption, infectious center formation, and single-cell virus production. BALB/c mouse embryo fibroblasts (MEF) produced more intracellular and extracellular virus than C57BL/6 MEF, with differences in virus production first appearing at 16 hr after inoculation. Virus yield in C57BL/6 cells peaked earlier (16 hr) and at a lower level than in BALB/c cells (20 hr). These results were explained by a difference in single-cell virus replication, rather than less efficient adsorption or the presence of cells that could not be infected. Host-related variation in the ability of infected cells to support HSV replication may account, in part, for differences in the severity of HSV ocular disease.

Laser densitometric analysis of class I HLA antigen expression by corneal epithelium.

Laser densitometric analysis of immunoperoxidase stained tissue was used to quantitate class I HLA (HLA-A,B,C) antigen expression by human corneal epithelium. Frozen sections of human donor corneas stored in modified McCarey-Kaufman medium for less than 24 hr were evaluated for class I HLA antigen by an indirect immunoperoxidase technique using a monoclonal antibody reactive against a class I HLA antigen determinant. Photomicrographs of stained epithelium taken under standardized conditions were evaluated by laser densitometry. Measurements from peripheral and central corneal epithelium on the same tissue section were compared. Total stain, stain density, and stain intensity were higher for peripheral than for central corneal epithelium, indicating that class I HLA antigen expression is greater for peripheral than for central epithelium.

Herpes simplex virus type 1 transcription is not detectable in quiescent human stromal keratitis by in situ hybridization.

PURPOSE: The purpose of this study was to determine whether herpes simplex virus (HSV) transcripts are present in the corneas of patients with chronic herpetic stromal keratitis. METHODS: Corneal buttons from patients with a history of stromal keratitis, but no ongoing active disease, together with positive and negative control tissues, were analyzed by in situ hybridization using single-stranded RNA probes for all three classes of viral lytic cycle transcripts as well as for the latency-associated transcripts (LATs). Tissues also were screened for presence of HSV genomic DNA using the polymerase chain reaction (PCR). RESULTS: HSV DNA was detected in 7 of 13 quiescent corneas by PCR, but no viral transcripts were detected in any of these corneas by in situ hybridization. CONCLUSIONS: At the level of detection afforded by in situ hybridization, HSV persistent in scarred human corneas after stromal keratitis appears to be transcriptionally dormant. This contrasts with the situation in neurons of latently infected sensory ganglia, in which LATs are present at high levels.

Corneal and conjunctival crystals in paraproteinemia.

A 64-year-old woman with bilateral corneal and conjunctival crystal deposition was evaluated. A biopsy of her conjunctiva showed intracytoplasmic inclusions of immunoglobulin crystals in fibrocytes, macrophages, and endothelial cells. Serum protein electrophoresis showed elevated kappa and IgA light and heavy chains which corresponded with immunoperoxidase staining results of the conjunctival biopsy. Conjunctival and corneal crystal deposition may be indicative of paraproteinemia, and histopathologic examination of a conjunctival biopsy may be useful in diagnosing this condition.

Penetrating keratoplasty for pseudophakic corneal edema with exchange of intraocular lenses.

We studied one eye in each of 25 consecutive patients with pseudophakic corneal edema. Each patient underwent a penetrating keratoplasty with exchange of intraocular lenses and was followed up for 12 to 38 months (mean, 18.7 months). The corneal graft remained clear in 22 (88%) eyes, but only eight eyes (32%) had acuity of 20/40 or better; nine eyes (36%) manifested cystoid macular edema, and six eyes (24%) had degenerative maculopathy. Elevated intraocular pressure was present in 12 eyes after surgery, with three eyes manifesting visual field loss; in all eyes the condition was controlled medically. Peripheral anterior synechiae appeared postoperatively in two eyes. We now treat severe cases of pseudophakic corneal edema with a penetrating keratoplasty, meticulous anterior vitrectomy, gonioplasty, iridoplasty, and exchange of intraocular lenses, employing a flexible-loop anterior chamber lens or a posterior chamber lens sutured to the iris.

Corneal thickness changes after high-risk penetrating keratoplasty. Collaborative Corneal Transplantation Study Group.

OBJECTIVE: To determine whether ultrasonic measurements of corneal thickness are of prognostic value after high-risk penetrating keratoplasty. DESIGN: A prospective, multicenter, randomized trial. PATIENTS: Four hundred fifty patients at high risk for graft failure because of previous immunologic graft failure or because of two or more quadrants of stromal vascularization. Patients underwent surgery and were treated according to a specific protocol and observed at frequent intervals. INTERVENTION: At each postoperative visit, ultrasonic measurement of central corneal thickness was performed and the corneal status was determined by biomicroscopic examination. MAIN OUTCOME MEASURE: Graft failure owing to immunologic or nonimmunologic causes. RESULTS: Corneal thickness stabilized by 3 months at a median thickness of 0.54 mm. The range of corneal thicknesses in patients with corneal grafts judged to be clear was large. In 49% of eyes, development of an allograft reaction episode was accompanied by an increase in corneal thickness of at least 10%; the greater the increase in thickness, the more likely the graft would fail. Clear grafts with central thicknesses of 0.59 mm or greater at 1, 3, or 6 months had a much greater risk of failure than those with thicknesses of less than 0.59 mm. CONCLUSION: Corneal thickness measurements after high-risk penetrating keratoplasty are of prognostic value.

Primary corneal graft failure. A national reporting system. Medical Advisory Board of the Eye Bank Association of America.

OBJECTIVES: To describe a national eye banking registry and to assess the effects of donor age, cause of donor death, time from death to procurement, storage time, and distance between the points of recovery and transplantation on the reported occurrence of primary corneal graft failure. DESIGN: We performed a retrospective case-control study to estimate the odds ratios of five donor factors for cases of primary graft failure voluntarily reported to a national registry using controls from selected eye banks. We also performed a nested case-control cohort study to compare cases of primary graft failure that occurred in both corneas from the same donor with those of nonmated corneas in which primary graft failure was reported to assess odds ratios for the same donor factors. PATIENTS: One hundred forty-seven patients developed primary graft failure in penetrating keratoplasty transplantations performed between January 1, 1991, and December 31, 1993. These cases were reported to the Adverse Reaction Registry of the Eye Bank Association of America, Washington, DC. Controls included 7240 donor corneas distributed by nine eye banks during 1992. RESULTS: Of the 147 donor corneas that developed primary graft failure, 17 (12%) were obtained from donors who were aged 70 years and older, 39 (27%) came from donors who died of trauma, 13 (9%) had a cadaver time longer than 12 hours, 10 (7%) had a storage time longer than 7 days, and 38 (26%) were distributed outside the eye bank's region. Compared with controls, these donor corneas were more likely to have a storage time longer than 7 days (odds ratio, 2.4; 95% confidence limits, 1.2 and 4.6) and to come from donors aged 70 years and older (odds ratio, 2.4; 95% confidence limits, 1.4 and 4.0). The 22 corneas (15%) in which primary graft failure occurred in both recipients from the same donor were 8.5 times (95% confidence limits, 1.1 and 51.5) more likely to be preserved beyond 1 week than were nonmated corneas with primary failure but were not from significantly older donors. Logistic regression analysis showed that the association between prolonged storage time and primary graft failure in mated corneas remained significant even when the analysis was controlled for other donor factors. CONCLUSIONS: No clearly defined donor or eye banking factor accounted for most cases of primary graft failure, although prolonged corneal storage and advanced donor age may increase its risk. Ophthalmologists are urged to report to their eye bank all cases of primary graft failure and other adverse events that might be attributable to donor eye tissue.

Centrifugal cytology of ocular fluids.

Centrifugal cytology is a new technique for the preparation of ocular fluids for cytologic examination. It differs from conventional cytocentrifuge techniques, since fixation is carried out simultaneously with centrifugation. It avoids air-drying artifacts and can be applied to small (50 microL) or large (200 mL or more) sample volumes and dilute cell suspensions, such as might be obtained by anterior chamber paracentesis, vitreous aspiration, or vitrectomy. The resultant preparation is a permanent, stained, well-preserved, flattened, homogeneous cell dispersion suitable for detailed cytologic evaluation.

Laser trabeculoplasty for glaucoma in aphakic and pseudophakic eyes after penetrating keratoplasty.

We used argon laser trabeculoplasty to treat medically uncontrolled glaucoma after penetrating keratoplasty in ten eyes that were aphakic or pseudophakic. These patients were examined over an average of 22.8 months (range, 12 to 37 months) after treatment. We found an average decrease in intraocular pressure of 9.1 mm Hg (range, +8 to -19), from an average of 30.6 mm Hg before treatment to an average of 21.5 mm Hg after treatment. Eight of ten eyes had a reduction of greater than 5 mm Hg, and six of ten eyes had intraocular pressure of less than 20 mm Hg. Visual function remained stable, and complications were rare. We now prefer argon laser trabeculoplasty to cyclocryotherapy for the initial treatment of patients with medically uncontrolled glaucoma who have predominantly open angles and a clear penetrating keratoplasty.

Iris melanoma seeding through a trabeculectomy site.

A 59-year-old man who had previously undergone a trabeculectomy in his right eye was examined because of an enlarging pigmented lesion of the inferior portion of the iris. A fine-needle aspiration biopsy of aqueous fluid revealed spindle cells and epithelioid malignant melanoma cells. The eye was enucleated, and subsequent histopathologic examination demonstrated a mixed spindle cell and epithelioid cell melanoma of the inferior portion of the iris with seeding of melanoma cells into the conjunctival filtering bleb via the trabeculectomy site. This case illustrates the usefulness of fine-needle aspiration biopsy in the evaluation of pigmented iris lesions and illustrates that iris melanoma can seed through a trabeculectomy site.