Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.

Growing vascular endothelial cells express somatostatin subtype 2 receptors.

We hypothesized that non-proliferating (quiescent) human vascular endothelial cells would not express somatostatin receptor subtype 2 (sst 2) and that this receptor would be expressed when the endothelial cells begin to grow. To test this hypothesis, placental veins were harvested from 6 human placentas and 2 mm vein disks were cultured in 0.3% fibrin gels. Morphometric analysis confirmed that 50-75% of cultured vein disks developed radial capillary growth within 15 days. Sst 2 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the RNA from veins before culture and from tissue-matched vein disks that exhibited an angiogenic response. The sst 2 gene was expressed in the proliferating angiogenic sprouts of human vascular endothelium. The presence of sst 2 receptors on proliferating angiogenic vessels was confirmed by immunohistochemical staining and in vivo scintigraphy. These results suggest that sst 2 may be a unique target for antiangiogenic therapy with sst 2 preferring somatostatin analogues conjugated to radioisotopes or cytotoxic agents.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Persistent infection of a lymphoma cell line by herpes simplex virus.

The peripheral blood cells from a patient with a B-cell lymphoma were established in long-term tissue culture. Two years after establishment of the cells in culture they were infected with herpes simplex virus type 2 and the productivity and duration of viral persistence investigated. One week after infection the lymphoblastoid cells were productively infected and have remained so for a period of over 3 years. Expression of a viral glycoprotein antigen was evaluated by using a fluorescein-labeled monoclonal anti-herpes simplex virus type 2 antibody and revealed a spectrum of staining reactions grading from a lightly stippled to very intense pattern. Polymerase chain reaction analysis of the infected cells revealed the presence of the herpes simplex virus type 2 DNA polymerase gene in the infected cells that was absent from the uninfected lymphoblastoid cells. These results taken together with the long-term growth characteristics of both the infected and uninfected lymphoblastoid cells suggest that this cell line may be a good model system for studying viral infection, viral replication, viral latency, and clinical application for the isolation of human herpes virus.

Reactivation of herpes virus after lamellar keratoplasty.

PURPOSE: To determine if lamellar keratoplasty in rabbits latently infected with herpes simplex virus type 1 (HSV-1) would stimulate graft recipients to shed virus and induce viral-specific corneal lesions. METHODS: Rabbits latently infected with HSV-1 received lamellar allografts in one eye from normal uninfected rabbits and the contralateral eyes served as unoperated controls. Normal rabbits received lamellar grafts from rabbits latently infected with HSV-1. For 1 week after surgery, slit-lamp examination and ocular swab sampling were performed daily to assess viral reactivation. RESULTS: The occurrence of positive swab cultures and corneal epithelial lesions after lamellar keratoplasty was significantly higher in operated eyes of latently infected rabbits when compared to the control eyes. Ocular shedding or recurrent lesions were not observed in the normal rabbits receiving corneal grafts from latently infected donors. CONCLUSIONS: These results indicated that lamellar keratoplasty induces HSV-1 shedding and recurrent epithelial lesions in the eyes of rabbits latently infected with HSV-1, which received lamellar grafts, but not in the eyes of normal rabbits given lamellar grafts from HSV-1 latently infected rabbits. It seems that the site of viral latency is not the anterior corneal stroma or the epithelium.

The relationship between interleukin-6 and herpes simplex virus type 1: implications for behavior and immunopathology.

Cytokines are hormones once thought to be restricted to the immune system produced solely by hematopoietic-derived cells and acting on receptors expressed by cells of the immune system. However, it is now clear that many cytokines are produced not only by lymphocytes, monocytes, granulocytes, and dendritic cells but are also synthesized by cells outside the realm of the immune system in response to stimuli that may not be associated with immune homeostasis. In fact, there is evidence supporting a role of selected cytokines modifying behavior and neuroendocrine function. Recently, a potential relationship between the cytokine interleukin (IL)-6 and herpes simplex virus type 1 (HSV-1) reactivation has been found. This article discusses the relevance of these findings and considers the potential impact that HSV-1 infection has on behavior and chronic inflammatory processes that can occur in the nervous system during "latent" virus infection as a result of chronic IL-6 expression.

Protection of corneal allografts by CTLA4-Ig.

PURPOSE: CTLA4, a high-affinity ligand of B7, can, in soluble form, prevent antigen-driven T-cell activation by blocking CD28-B7 interaction and can thereby prevent immune graft rejection. In this study, we tested the capacity of soluble CTLA4-Ig alone or in combination with UV-B irradiation to suppress corneal allograft rejection in rabbits. METHODS: Corneas from Dutch belted rabbits were incubated in corneal storage medium containing 0, 1, 10, 25, or 250 microg/ml of CTLA4-Ig for 18 h and were then transplanted into the vascularized or nonvascularized corneas of New Zealand White rabbit recipients. A series of donor corneas were exposed to UV-B irradiation alone or a combination of irradiation and CTLA4-Ig to determine if these two treatments would have an additive effect in prolonging graft survival. The fate and clinical condition of the allografts were evaluated by slit-lamp photomicroscopic observation and corneal-thickness measurements. Grafts that were rejected were processed for histopathologic and immunohistochemical analysis to determine the characteristics of cells infiltrating the grafts. RESULTS: Grafts placed in nonvascularized corneas showed no differences in survival times, regardless of treatment. Among the grafts placed in vascularized corneas, those incubated with CTLA4-Ig at a concentration of 250 microg/ml failed within 7-14 days. Histopathologic and immunocytochemical examination revealed a dense accumulation of immune inflammatory cells, especially class II major histocompatibility complex (MHC)-expressing, antigen-presenting cells, in the failed grafts. Grafts incubated with CTLA4-Ig at concentrations of 1 and 10 microg/ml had mean survival times greater than the control, untreated corneal allografts. Some of the grafts in these two treatment groups survived for the 100-day observation period, whereas none of the grafts in the other treatment groups survived to this end point. UV-B irradiated grafts incubated with CTLA4-Ig at a concentration of 1 microg/ml appeared to have longer survival times and fewer rejections compared with control, untreated grafts and grafts treated with UV-B or CTLA4-Ig alone. CONCLUSION: The results show that the CTLA4-Ig coreceptor blocking agent can prolong corneal allograft survival in vascularized graft sites and that UV-B irradiation followed by incubation in CTLA4-Ig may prolong graft survival better than either treatment alone. These results suggest that agents that prevent second-signal interaction between antigen-presenting cells and T lymphocytes may be useful for inhibiting corneal allograft rejection.

Acyclovir blocks cytokine gene expression in trigeminal ganglia latently infected with herpes simplex virus type 1.

We have previously found that interleukin (IL)-2, IL-10, interferon (IFN)-gamma, RANTES, and tumor necrosis factor (TNF)-alpha mRNA transcription remain elevated in the trigeminal ganglia (TG) of herpes simplex virus type 1 (HSV-1) latently infected mice up to 120 days postinoculation (p.i.). To determine if this phenomenon was dependent on HSV-1 DNA replication after the establishment of latency (i.e., reactivation), cytokine gene expression was compared in TG of acyclovir-treated and untreated latently infected mice. Oral acyclovir treatment (begun 16 days p.i.) had no effect on serum levels of total anti-HSV-1 antibodies. However, there was a significant reduction in the titer of antibody specific for glycoprotein D and glycoprotein B but not glycoprotein H/L 120 days PI in the acyclovir-treated compared to vehicle-treated mice. These differences were not significant at earlier time points (i.e., days 34 and 60 p.i.). Consistent with these findings, acyclovir had no effect on cytokine gene expression in latently infected TG 35 and 60 days p.i. However, 120 days p.i., IFN-gamma and TNF-alpha mRNA were approaching baseline levels in TG of acyclovir-treated mice, but remained significantly elevated in untreated controls (i.e., IFN-gamma mRNA levels were sixfold higher in TG of untreated mice). Therefore, viral DNA replication appears to provide an antigenic stimulus for persistent cytokine gene expression in latently infected TG.

Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay.

PURPOSE: To determine whether substance P is present in human tears. METHODS: Tear samples (1-2 microliters) were collected from one eye of each of 12 subjects. Two of the eyes had dry eye syndrome, two wore contact lenses and had dry eye syndrome, and eight were normal. Five of the eight normal eyes were scheduled to undergo excimer laser refractive surgery, and tears were collected from these eyes before and after surgery. Tear samples were analyzed by laser desorption mass spectrometry. Pooled samples from one individual were subjected to enzyme-linked immunoabsorbent assay. RESULTS: Laser desorption mass spectra of the 18 tear samples displayed well defined peaks with mass to charge (m/z) ratios ranging from 1343.7 to 1355.9 and/or 1356.9 to 1364.7, corresponding to an average m/z of 1349.8 +/- 1.13 for protonated substance P and 1361.2 +/- 0.54 for oxidized substance P obtained from 14 mass spectra of standards formulated with substance P concentrations ranging from 10(-4) M to 10(-12) M. As confirmation, an enzyme-linked immunoabsorbent assay performed twice on pooled tears from one eye detected substance P in both replicates at a concentration of 125 pg/ml (9.26 x 10(-11) M). CONCLUSIONS: These findings demonstrate that substance P is a component of tears obtained from normal eyes of men and women ranging in age from 26 to 60 years, from eyes fitted with contact lenses, from eyes with dry eye syndrome, and from eyes 1 and 2 days after excimer laser refractive surgery. Whether the concentration of substance P in tears varies with sex, age, or eye condition, the source of substance P in tears, and its role in tears remains to be discovered.

Breast cancer increases initiation of angiogenesis without accelerating neovessel growth rate.

BACKGROUND: Recurrence and mortality rates in patients with breast cancer correlate with the degree of tumor angiogenesis (angiogenic index). We have developed a novel angiogenesis model by using disks of fresh human placental vein that initiate an angiogenic response and exhibit linear radial capillary growth in culture. We hypothesized that the addition of human breast cancer cells to this human placental vein angiogenesis model would increase the incidence of angiogenesis and accelerate the rate of neovessel growth compared with vein disk cultured without tumor cells. METHODS: To test this hypothesis, vein explants from seven human placentas were incorporated into clots of 0.3% fibrin in Medium 199 and fetal bovine serum with or without 1.5 x 10(5) T-47D (n = 6 placentas) or MCF-7 (n = 1 placenta) breast cancer cells. Statistical differences between the experimental (with breast cancer cells) and control (no added cells) cultures were determined by repeated measures ANOVA. RESULTS: The proportion of disks exhibiting neovessel growth (initiation) by day 12 was significantly increased in the presence of T-47D cells (p < 0.05 at day 12, p < 0.001 at day 15). No statistical difference was seen in rates of neovessel growth (millimeters per day). Similar results were seen with MCF-7 cells. CONCLUSIONS: Tumor enhancement of angiogenesis may occur by increased initiation of the angiogenic response. Subsequent vessel growth rates may be tumor independent. We predict that effective antiangiogenic therapies will block a tumor's ability to augment angiogenesis initiation rather than subsequent neovessel growth.

Anti-interleukin-6 antibodies inhibit herpes simplex virus reactivation.

Herpes simplex viruses (HSVs) infect epithelial cells, become localized in neurons, and can reactivate in response to a variety of stimuli, including ultraviolet light and hyperthermia. The sequence of gene activation during viral replication is known, but the molecular linkage between exogenous stimuli and HSV reactivation has not been determined. It was hypothesized that interleukin (IL)-6 acts as a signal between exogenous stimuli and neurons, stimulating HSV reactivation from latency. Mouse corneas were infected with HSV-1, and ocular reactivation was induced 5-7 weeks later by thermal stress or corneal exposure to ultraviolet light. Anti-IL-6 monoclonal antibodies were administered to the latently infected mice 8-12 h before the reactivation stimulus. Treatment with anti-IL-6 antibodies resulted in significantly lower frequencies of ocular reactivation compared with those in mice treated with a control immunoglobulin. These results support the hypothesis that IL-6 plays a role in HSV reactivation from latency.

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.

Following ocular infection, herpes simplex virus type 1 (HSV-1) establishes latency in trigeminal ganglion (TG) neurons. Using reverse transcription-PCR, cytokine gene expression was analyzed in the TGs of mice infected with HSV-1. IL-2, TNF-alpha, IFN-gamma, IL-10, and RANTES mRNAs were readily detected in TGs taken from mice 7 days postinoculation (PI). Likewise, IL-2, IL-6, IL-10, and IFN-gamma protein were detected by ELISA of TG homogenates. Between 5 and 45 days PI, IL-10, IFN-gamma, TNF-alpha, and RANTES mRNAs were detected in nearly 100% of latently infected TGs (latent infection was confirmed by reverse transcription-PCR detection of HSV-1 latency-associated transcripts). T cell-associated cytokine and chemokine mRNAs (IL-2, IL-10, IFN-gamma, and RANTES) were still detected in the majority of latently infected TG samples taken between 60 and 135 days PI. In contrast, these cytokine mRNA species were rarely detected in uninfected TGs. Measurement of serum Abs to HSV-1 at different times revealed that anti-HSV-1 Ab concentrations approached a plateau in mice by 30 days PI but remained at high levels 67 and 125 days PI. Although there was molecular evidence of an ongoing immune response to HSV-1 in latently infected TG, histologic analysis indicated that very few mononuclear cells remained in the ganglion 60 days PI. Collectively, the results suggest that residual lymphocytes encounter viral Ag during HSV-1 latency with sufficient frequency to remain activated. The paradox of a persistent immune response against a latent infection is discussed.

Synthetic scleral reinforcement materials. II. Collagen types in the fibrous capsule.

Attainment of a steady state in fibrous-capsule formation around a polymeric implant is indicative of minimization of the foreign-body response, and is characterized by thin capsule walls, few macrophages, and the replacement of type III collagen with type I collagen within the capsular matrix. We implanted four general types of extraocular band materials (porous, solid, composite, and patched) in rabbit eyes, and examined the proportions of types III and I collagen in the surrounding fibrous-capsule walls at intervals after surgery. Immunohistochemical analysis showed that 18 months after implantation of the band materials, the capsules adjacent to the porous-material surfaces had virtually no type III collagen remaining, while the solid-surface-material capsules still contained more than trace amounts (greater than 5%). In that the bands were implanted around the outside of the globe, which provides a dynamic environment in constant motion, it may be that the cellular ingrowth permitted by the porous materials increased the stability of the implants, thereby lessening the foreign body response and allowing the collagen replacement characteristic of the steady state to be completed. In contrast the solid-surface implants, which had little cellular ingrowth, may have continued to undergo sufficient micromovement within the capsule walls to sustain a low-level activation of the wound-healing response, resulting in thicker capsule walls and residual type III collagen a year and a half after implantation surgery.

Chronic morphine treatment suppresses CTL-mediated cytolysis, granulation, and cAMP responses to alloantigen.

Exposure to opioid drugs (e.g., morphine) in vivo has been shown to suppress natural killer cell activity. However, the effects of in vivo exposure to opioids on cytotoxic T lymphocyte (CTL) activity has not been investigated. The administration of morphine (50.0 mg/kg, sc) to alloimmunized mice for 11 days resulted in a significant decrease in peritoneal and splenic CTL activity. Moreover, the intracellular content of serine esterases and esterase release by CD8+ effector cells from chronic morphine-treated mice was reduced compared to that of effector cells from vehicle-treated controls. In addition, the CD8+ cAMP response to alloantigen was diminished compared to CD(8+)-enriched cells from vehicle-treated animals. However, conjugate formation between effector and target and subsequent killing of target by effector cells did not reveal significant differences between vehicle- and chronic morphine-treated animals. Serum corticosterone and dehydroepiandrosterone levels were significantly lower in the chronic morphine-treated animals while proopiomelanocortin gene expression (exon 3) in splenic lymphocytes did not correlate with morphine-mediated suppression of CTL activity. These results indicate that CTL activity is sensitive to chronic morphine exposure, implicating opioids as important cofactors during viral infections in suppressing cell-mediated immunity.

Central alpha-adrenergic involvement in morphine-mediated suppression of splenic natural killer activity.

Alpha 1-adrenergic pathways are involved in morphine-induced suppression of murine splenic NK activity. To investigate the level of involvement following morphine administration, the peripheral acting alpha-adrenoceptor antagonist doxazosin and the broad acting alpha-adrenoceptor antagonist phentolamine were employed. Mice preadministered phentolamine (2.0 mg/kg) exhibited a modest but insignificant suppression of splenic NK activity following morphine administration while mice preadministered doxazosin (1.0 mg/kg) or vehicle showed a significant decrease in splenic NK activity following morphine administration. Morphine was also found to significantly (P < 0.01) increase splenic serotonin levels (14.88 +/- 1.62 ng/mg) relative to saline-treated controls (7.3 +/- 0.9 ng/mg). Both phentolamine and doxazosin pretreatment completely or partially blocked morphine-mediated elevation of splenic serotonin levels, respectively. Morphine administration decreased the ability of NK cells to form conjugates with target (YAC-1 lymphoma) cells and decreased the number of active killer cells within the conjugate population. Collectively, these results implicate central alpha-adrenergic involvement following acute morphine administration in suppressing splenic NK activity indirectly through a reduction in the number of effector-target conjugates and active cytolytic effector cells.

A platelet-activating factor antagonist reduces corneal allograft inflammation and neovascularization.

We assessed the role of platelet-activating factor (PAF) in corneal allograft rejection and evaluated the effects of a PAF antagonist on corneal inflammation, cellular infiltration, vascularization, and edema. Rabbits with vascularized corneas served as recipients of allogeneic cornea grafts. Rabbits with normal corneas underwent autografts as controls. All of the allografts developed the progression of signs characteristic of rejection. Nevertheless, treatment with the PAF antagonist BN52021 significantly inhibited corneal allograft vascularization for up to 10 days after transplantation and reduced the number of eosinophils in the allografts at 28 days after transplantation. In contrast, saline-treated allografts exhibited florid vascularization and intense inflammatory infiltrates. Control autografts survived without developing significant inflammation or vascularization. The retardation of allograft eosinophilia and graft vascularization by the PAF antagonist was most likely the result of suppression of PAF-mediated reactions in the cornea. These results indicate that PAF may play a role in corneal inflammation and vascularization after corneal transplantation, and that PAF antagonists may be clinically useful in delaying some of the pathophysiologic consequences of corneal graft rejection.

Immunogenicity of epikeratophakia tissue lenses containing living donor keratocytes.

The purpose of this study was to compare the survival of epikeratophakia tissue lenses prepared with cryolathing and lyophilization (frozen lenses) and without (fresh lenses) in donor-sensitized recipients with vascularized corneas. Fresh lenses placed in vascularized corneas of immune recipients were subjected to immune attack. Frozen lenses placed in vascularized corneas of immunized recipients did not elicit an immune reaction. Neither fresh nor frozen lenses elicited immune reactions in nonvascularized corneas of immune recipients or in nonvascularized, nonimmune recipients. These results indicate that although the fresh lenses are more antigenic than the lenses in which the cells have been killed by freezing and lyophilization, the fresh lenses prepared using the BKS-1000 technique containing living stromal keratocytes are not likely to stimulate allograft immune reactions in unsensitized patients with avascular graft sites.

The role of class II antigen-expressing cells in corneal allograft immunity.

The goal of this study was to investigate the relationship between the presence of Class II antigen-expressing cells (Class II+) cells in the cornea and the generation of allograft immunity. Wistar/Furth (W/F) rat Class II+ cells were injected in various numbers (0.01, 0.1, 1.0, 5.0, 10.0, and 20.0 X 10(6) into the corneal stroma of Fischer 344 (F344) rats. For comparison, the same numbers of W/F Class II+ cells were injected directly into the peritoneal cavity of F344 rats. Also, W/F cells were injected into the corneas of F344 rats and the corneas were "grafted" intraperitoneally in F344 hosts. The results showed that up to 20 X 10(6) class II+ cells injected in situ into the corneal stroma did not elicit a serum cytotoxic antibody response or a splenic or blood cytotoxic T-cell response against donor Class II antigens. In contrast, systemic immune responses were elicited by both direct intraperitoneal injection of large numbers (10 or 20 X 10(6] of allogeneic Class II+ cells and by intraperitoneal grafting of syngeneic corneas carrying similar numbers of allogeneic Class II+ cells. F344 recipients of a syngeneic cornea containing 10 or 20 x 10(6) W/F Class II+ cells or a suspension of W/F cells exhibited an accelerated rejection of W/F skin allografts (13.3 versus 9.0 days, first- versus second-set rejection). These results indicate that the number of Class II+ cells required to elicit a systemic immune response is larger than the number of Class II+ cells present in the normal cornea.