Barriers to Emergency Department Utilization of AFIB Protocol in Uncomplicated Lone AFIB Patients-Results from an Online Survey Study.

BACKGROUND: Historically, atrial fibrillation (AFIB) management has focused on rate control and anticoagulation, necessitating hospital admission. Recently, some emergency departments (EDs) have implemented protocols to avoid hospital admission when managing lone AFIB. Despite this recent trend, there is still reluctance toward the implementation of these protocols by some emergency physicians (EPs). OBJECTIVE: This study investigates barriers to implementation of ED AFIB protocols by surveying which aspects may impede their use. METHODS: To analyze the perceived barriers from EPs, we formulated a survey assessing the various components of ED AFIB management to identify which aspects might impede EP utilization. It was distributed as an email to large national ED physician group. Data was analyzed using descriptive means and weighted averages. RESULTS: Of 185 respondents (response rate 6.1%), 17.4% already had AFIB protocols in place at their home institutions and 82.6% did not. Majority opinion of largest barriers toward the implementation of AFIB protocols were the extended ED length of stay and discharge with unclear follow-up. There was little concern with chemical and electrical cardioversion and very limited concern with rate control and initiating oral anticoagulation. EPs supported placement in Observation for implementation and involvement of discharge planning to establish prescriptions and follow-up. CONCLUSION: EP input regarding the development of ED AFib protocols will be essential in order to develop cost effective, convenient and safe methods of treatment. This survey of EP suggests that ED length of stay and insuring close outpatient follow up are key issues to address as protocols are designed.

Role of the immune system in mediating the antitumor effect of benzophenothiazine photodynamic therapy.

The role of the host immune system in contributing to tumor regression following benzophenothiazine photodynamic therapy (PDT) was examined. Photodynamic therapy with 2-iodo-5-ethylamino-9-diethylaminobenzo[a]-phenothiazinium chloride (2I-EtNBS) eradicated EMT-6 mammary fibrosarcomas in 75-100% of treated mice. In contrast, PDT failed to inhibit tumor growth in T-cell-deficient nude mice. Furthermore, T-cell depletion studies with anti-CD8 antibody revealed that the CD8+ T-cell population was critical for an effective PDT response (tumor volume 14 days post-PDT: 262 mm3 vs 59 mm3 in controls; P < 0.01). Because anti-CD4 antibody inhibited tumor growth in the absence of PDT, the role of CD4+ T cells remains unclear. Depletion of natural killer (NK) cells in vivo with anti-asialo-GM1 antibody significantly reduced a suboptimal PDT effect relative to vehicle controls (tumor volume 13 days post-PDT: 513 mm3 vs 85 mm3, respectively; P < 0.001). However, splenic NK cells obtained from PDT-treated tumor-bearing mice were not cytotoxic in vitro against EMT-6 cells, suggesting that NK cells contribute to the PDT effect in vivo by an indirect mechanism. In addition, when mice with complete tumor regression following PDT were rechallenged 28 days later with 5 x 10(5) EMT-6 cells, tumor growth was significantly inhibited as compared to controls (tumor volume 40 days postrechallenge: 137 mm3 vs 833 mm3 in controls; P < 0.03; percent animals without tumor in five experiments: 67% vs 8% in controls). Collectively, these results demonstrate that CD8+ T cells are required to prevent tumor regrowth after 2I-EtNBS-PDT, NK cells contribute to this response and such PDT can elicit protective antitumor immunity.

The immunoregulatory effects of prolactin in mice are time of day dependent.

The effects of timed administration of PRL on immune activities were investigated in male BALB/c mice. Ten daily injections of PRL (1 mg/kg) were made 0/24, 4, 8, 12, 16, or 20 h after light onset (HALO). On day 11, spleen cells were harvested between 1-3 HALO and cocultured with gamma-irradiated C57BL/6 spleen cells for 5 days, and proliferative responses to alloantigen were assayed (mixed lymphocyte reaction). When given in vivo at 4-12 HALO, PRL strongly stimulated proliferation by more than 2-fold, whereas PRL injections when given at 24 HALO substantially inhibited proliferation and had no effect when given at 16-20 HALO. When endogenous PRL secretion was stimulated for 7 days with injections of domperidone or 5-hydroxytryptophan, the splenocyte response increased by 48% and 64%, respectively, when injections were given at 9-10 HALO, but did not increase when they were given at 23-0 HALO. Inhibition of endogenous PRL secretion for 7 days with bromocriptine (2.5 mg/kg.day) inhibited splenocyte responsiveness by 40% when injected at 9 HALO, but had no effect when administered at 0 HALO. Furthermore, such bromocriptine treatment inhibited T- and B-cell mitogenic responses to Concanavalin-A (by 48%) and lipopolysaccharide (38%) when administered at 10, but not 0, HALO. In a manner similar to mixed lymphocyte reaction responses, daily PRL injections for 10 days at 11 HALO stimulated (40%) the in vivo delayed-type hypersensitivity response to antigen (azobenzenearsonate), whereas injections at 0 HALO were nonstimulatory. Bromocriptine treatment (1.5 mg/kg.day) suppressed the delayed-type hypersensitivity response (43% less than the control value) when administered at 10-12 HALO, but had no effect when administered at light onset. Timed PRL injections for 28 days in adult mice increased (42%) the total thymic cell number when administered at 11 HALO, but had no effect when injected at 0 HALO. Together, these results show that immunocyte responsiveness to PRL is time of day dependent. Thus, these findings support an essential and heretofore unrecognized circadian role in PRL regulation of immunity.

Inhibition of development of murine melanoma lung metastases by systemic administration of recombinant platelet factor 4.

BACKGROUND: When administered locally, recombinant platelet factor 4 (rPF4), a known angiogenesis inhibitor, has been shown to effectively suppress murine melanoma and human colon carcinoma primary tumor growth in mice. It was tentatively concluded that this effect was due to the inhibition of tumor neovascularization. PURPOSE: This study has evaluated the effects of systemically administered rPF4 on the growth and establishment of experimental B16F10 melanoma lung metastases in syngeneic mice. METHODS: B16F10 cells (0.5-1.0 x 10(5) were administered intravenously to mice; 21 days later, the lungs were removed and the tumor foci were counted. Treatments with rPF4 were given to C57BL/6J mice immediately following tumor inoculation either (a) intravenously as a single dose (0.375, 0.75, 1.5, or 2.0 mg) or as multiple doses (6 mg total) over a 48-hour period, (b) subcutaneously or intramuscularly as multiple doses (6 mg total) over a 72-hour period, or (c) subcutaneously as multiple doses (6 mg total) over a 92- or 96-hour period following a delay of 4, 24, or 48 hours. (BALB/cByJ x C57BL/6)F1 (CByB6F1/J) athymic nude mice received rPF4 subcutaneously over a 72-hour period. The ability of rPF4 to block binding of 51Cr-labeled B16F10 tumor cells to matrix-coated microtiter plates was evaluated in vitro. The in vivo effect of intravenously injected rPF4 on the retention of 51Cr-labeled B16F10 cells was examined by determining the remaining lung-associated radioactivity after 30 minutes or 1, 2, or 4 hours. RESULTS: Intravenous administration of rPF4 significantly inhibited the development of metastatic lung nodules in a dose-dependent fashion as assessed by both lesion number (P < .03) and lung weight (P < .05). When initiation of subcutaneous treatment with rPF4 was delayed 24-48 hours, the number of metastatic foci was significantly reduced (P < .05). The antitumor effect of rPF4 was not dependent on a T-lymphocyte-mediated process, since subcutaneous rPF4 treatment also suppressed the number of lung metastases in T-cell-deficient athymic CByB6F1/J nude mice. In vitro experiments demonstrated modest inhibitory effects (28%) of rPF4 on B16F10 tumor cell binding to purified murine vitronectin. However, lung clearance experiments at early time points (0.5 hour and 1 hour) showed that tumor lodging was not affected by rPF4 treatment. CONCLUSIONS: The administration of rPF4 by systemic routes inhibited the development of experimental lung metastases. These findings are consistent with the known angiostatic properties of rPF4, and we conclude that inhibition of metastatic tumor formation may be due to inhibition of tumor-induced neovascularization. IMPLICATIONS: These results support the testing of rPF4 as an angiostatic agent in the treatment of metastatic tumors.

Production of interleukin-6 and granulocyte-macrophage colony-stimulating factor by murine iris and ciliary body explants.

PURPOSE: To examine the ability of murine iris-ciliary body explants to produce cytokines with proinflammatory activities. METHODS: Supernatants derived from murine iris-ciliary body (I-CB) tissue explants cultured (four per well in 1 ml medium) in the presence of indomethacin were analyzed for the production of IL-1, IL-2, IL-3, IL-6, tumor necrosis factor-alpha/beta (TNF alpha/beta) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Analysis was performed by thymocyte costimulation, growth factor-dependent cell proliferation, TGF-beta-sensitive mink lung epithelial cell proliferation, and enzyme-linked immunosorbent assays (ELISA). RESULTS: Supernatants from I-CB explants cultured in vitro for 24 hours contained significant thymocyte costimulatory activity. This activity was fully neutralized by a combination of antisera to IL-1 and IL-6, and ELISA analysis confirmed that IL-6 was a significant component of the supernatant (402.7 pg/ml). TNF alpha/beta were also found in low concentrations (approximately 2.0 U/ml) by ELISA analysis, whereas IL-2 and IL-4 were not detectable. Significant amounts of GM-CSF (15.8 U/ml), but no IL-3, were detected. CONCLUSIONS: These results demonstrate that normal I-CB tissue contains cells capable of producing IL-6, GM-CSF, and IL-1. Because of the proinflammatory nature of IL-6 and IL-1, and the ability of GM-CSF, IL-1, and IL-6 to enhance functional capabilities of antigen presenting cells, regulation of the production of these cytokines may contribute significantly to the maintenance of the immunologic status of this regional site.

The presence of biologically significant concentrations of glucocorticoids but little or no cortisol binding globulin within aqueous humor: relevance to immune privilege in the anterior chamber of the eye.

PURPOSE: Immunosuppressive factors in aqueous humor (AH) contribute to the immune-privileged status of the anterior chamber of the eye. One such factor is transforming growth factor-beta (TGF-beta); other relevant inhibitors have not been fully identified. The authors examined AH to search for other putative inhibitors and to determine their effect on TGF-beta inhibitory activity. METHODS: Radioimmunoassays (RIA) were used to detect the presence of hydrocortisone, corticosterone, cortisol binding globulin (CBG), and alpha-melanocyte stimulating hormone (alpha-MSH) in AH. The ability of these factors to inhibit murine thymocyte proliferation stimulated by phytohemagglutinin-interleukin 1 (PHA/IL-1) and proliferation of a TGF-beta-sensitive cell line (CCL64) in vitro was examined. The ability of hydrocortisone to inhibit a one-way mixed lymphocyte reaction (MLR) and the ability of epidermal cells to present soluble tumor-associated antigens (TAA) for elicitation of immunity in mice in the concentration range present in AH was also examined. RESULTS: Hydrocortisone was detected in mouse, rat, and human AH (10.8 +/- 1.1 ng/ml, 9.3 +/- 2.1 ng/ml, and 18.0 +/- 1.0 ng/ml, respectively; mean +/- SEM), as was corticosterone (2.7 +/- 0.9 ng/ml, 2.2 +/- 0.3 ng/ml, and 0.7 +/- 0.1 ng/ml, respectively). Whereas normal plasma contains a binding protein for corticosteroids (i.e., CBG), the concentration in mouse, rat, and human AH was less than the level detectable by an RIA. Hydrocortisone inhibited PHA/IL-1-stimulated murine thymocyte proliferation and CCL64 cell proliferation in the concentration range present in AH. When hydrocortisone was combined with TGF-beta 2 (125 pg/ml), the degree of inhibition observed was greater than with either alone. Corticosterone inhibited thymocyte costimulation only slightly at concentrations present in AH but was inhibitory for CCL64 cells. alpha-MSH was also detected in AH. The concentration present had only slight inhibitory effects for CCL64 cell proliferation and did not enhance TGF-beta 2-mediated (62 pg/ml to 250 pg/ml) inhibition of CCL64 or thymocyte proliferation. Hydrocortisone inhibited the one-way MLR in the concentration range present in AH and, at 10 ng/ml, inhibited the ability of epidermal cells to present TAA for elicitation of delayed-type hypersensitivity in tumor-immune mice. CONCLUSIONS: These results show that AH contains biologically relevant concentrations of glucocorticoids and that CBG is relatively absent so that glucocorticoids present are largely free, and they suggest that regional sites take advantage of the activities of multiple factors to maintain an immune-privileged status.

Aqueous humor inhibits epidermal cell antigen-presenting function.

Aqueous humor (AH) is a complex biological fluid containing factors that mediate a number of activities that may contribute to immune privilege in the anterior chamber of the eye. To determine whether AH may inhibit dendritic cell antigen-presenting function, we evaluated the ability of AH to alter the ability of freshly obtained epidermal cell (EC) preparations enriched for Langerhans cells (LC) to present tumor-associated antigens (TAA) derived from the S1509a spindle cell tumor for elicitation of a delayed-type hypersensitivity (DTH) reaction in tumor-immune mice. Fresh EC preparations contain 2-5% LC. Exposure of EC to media containing AH for 2 hours prior to TAA-pulsing and injection into a hind footpad of tumor-immune mice resulted in a dose-dependent decrease in the DTH response. Aqueous humor contains transforming growth factor-beta (TGF beta), primarily TGF beta 2 as determined by specific neutralization of activity in the Mv 1 Lu cell assay. However, exposure of EC to TGF beta 2 in this fashion prior to TAA-pulsing had no effect on the degree of hypersensitivity elicited. Furthermore, preincubation of AH with neutralizing antiserum to TGF beta 2 had no effect on the ability of AH to inhibit LC antigen-presenting function. Mixing of AH-exposed non-TAA-pulsed EC with TAA-pulsed EC not exposed to AH prior to footpad challenge did not diminish the DTH response, suggesting that carryover of AH into the footpad was not responsible for the inhibition observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of latent transforming growth factor-beta and other inhibitory factors by cultured murine iris and ciliary body cells.

Aqueous humor contains transforming growth factor-beta (TGF-beta) and other inhibitory factors for cellular proliferation. In the present study we investigated the possibility that these factors are produced locally by cells found within the iris and ciliary body. Iris and ciliary body (I/CB) cells or whole tissue explants from C57BL/6 mice produced soluble factors which inhibited both murine thymocyte and mink lung epithelial cell proliferation. Indomethacin partially blocked inhibition of thymocyte proliferation, but did not affect inhibition of Mv1 Lu proliferation. The inhibitory activity of culture supernatants was not blocked by neutralizing antibodies to TGF-beta 1 or TGF-beta 2. However, following acid activation of culture supernatants from both I/CB and corneal tissue, increased inhibitory activity consistent with activation of latent TGB-beta was detected. Antibody neutralization experiments demonstrated that this increase in activity was due primarily to TGF-beta 2 for I/CB tissue. Polymerase chain reaction (PCR) analysis of cDNA generated from I/CB tissue mRNA showed prominent fragments representing both TGF-beta 1 and TGF-beta 2 mRNA. Corneal tissue, however, showed a prominent fragment for TGF-beta 1 mRNA, but either no band or a barely detectable fragment for TGF-beta 2 mRNA. Therefore, it remains uncertain whether TGF-beta 2 mRNA is produced by the cornea in this strain. Overall, these results demonstrated that three distinct categories of substances inhibitory to proliferation may be locally produced by resident iris and ciliary body cells: 1) indomethacin sensitive products, 2) TGF-beta 2 in latent form, and 3) factors not blocked by indomethacin or anti-TGF-beta neutralizing antibodies. Products generated by these tissues may have important regulatory properties in the development of immune responses to antigens introduced into the anterior chamber.

Regulation of GM-CSF and IL-3 production from the murine keratinocyte cell line PAM 212 following exposure to ultraviolet radiation.

Ultraviolet radiation (UVR) exposure induces profound changes in the synthesis and secretion of various cytokines both in vivo and in vitro. Little is known regarding the mechanism of these responses. This investigation evaluated the effects of UVR on the ability of a murine keratinocyte line (PAM 212) to produce interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). Subconfluent rapidly dividing PAM 212 cells were shown by RNA slot-blot hybridization studies to have increased levels of mRNA for both IL-3 and GM-CSF within 1 h of UVR exposure. However, only GM-CSF-specific bioactivity, as determined by antibody neutralization studies, was shown to increase above baseline in cell supernatants. Cells grown to confluence responded differently to UVR. Under these culture conditions an apparent decrease in bioactivity was detected after UVR exposure for both growth factors, and no change in mRNA levels was detected. In addition to culture density, removal of extracellular calcium or sodium during irradiation, treatment with amiloride, or inhibition of new mRNA synthesis with cordycepin was shown to influence the UVR-induced alteration in release of IL-3 or GM-CSF bioactivity from both confluent and subconfluent PAM 212 cells. These results demonstrate that UVR influences the release of the colony stimulating factors GM-CSF and IL-3 from keratinocyte, and suggests that the state of cell growth and conditions of membrane ion transport influence the mechanisms regulating secretion of those factors.

Morphologic and ultrastructural examination of I-A+ cells in the murine iris.

The surface membrane expression of major histocompatibility (MHC) class II antigens is an important prerequisite for presentation of foreign antigens to the immune system. Because particular antigens that are placed within the anterior chamber of the eye elicit a deviant form of immunity in which effector delayed-type hypersensitivity responses are suppressed, it has been proposed that novel MHC class II antigen-bearing cells exist in the tissues that line the anterior chamber. Class II MHC antigen expression has been identified within the iris, but the detailed morphologic description of these cells is incomplete. With the use of in situ immunoperoxidase and immunoelectron microscopic techniques, we examined the morphologic and ultrastructural characteristics of resident MHC-positive class II (I-A+) cells in murine irises. A significant number of these cells was found in the connective tissue of BALB/c irises. The majority showed extensive dendritic morphologic characteristics and formed a network throughout the iris that did not overlap. Ultrastructurally, I-A+ cells had an indented nucleus, some vacuoles, lysosomes, mitochondria, and an occasional phagosome within their cytoplasm and an absence of desmosomes or other intercellular junctions. Based on these features, it is unlikely that these cells are epithelial or endothelial in origin, but rather are similar to cells of the monocyte/macrophage/dendritic cell lineage. These results show the presence of an I-A+ dendritic cell population, within the murine iris, distributed in a pattern that is similar to that of Langerhans cells in the skin. Due to their compartmentalization within the eye, this cell population may represent a novel antigen-presenting cell that contributes to the immunologic privilege of the anterior chamber.

Tumor antigen presentation by murine epidermal cells.

The ability of epidermal Langerhans cells to present Ag for CD4-dependent immunity is well documented, and it has been hypothesized that Langerhans cells participate in the generation of immunity against incipient epidermal neoplasms by presentation of tumor-associated Ag in situ. This study examined the ability of murine epidermal cells (EC) to present tumor-associated Ag for the induction of in vivo antitumor immunity. Murine epidermal cells were deleted of Thy-1-bearing cells, cultured in 50 U/ml granulocyte-macrophage-CSF for 14 to 18 h, and pulsed with tumor fragments (TF) derived from S1509a-fibrosarcoma cells. These TF-pulsed EC were injected s.c. into syngeneic recipients at weekly intervals for a total of three immunizations and challenged with viable S1509a tumor cells 1 wk after the last immunization. Control animals received TF-pulsed allogeneic EC or EC treated identically but not pulsed with TF. EC that were pulsed with tumor cell fragments were able to induce protective immunity to tumor growth in vivo and to immunize for a significant delayed-type hypersensitivity response to injected tumor cells. The induction of antitumor immunity with TF-pulsed EC was genetically restricted, and culture of EC in granulocyte-macrophage-CSF was required for development of significant immunity. Furthermore, deletion of I-A+ cells by antibody and complement-mediated lysis eliminated the generation of immunity. Thus, I-A+ epidermal cells are capable of presenting S1509a tumor Ag for the generation of protective antitumor immunity in vivo.

Aqueous humor contains transforming growth factor-beta and a small (less than 3500 daltons) inhibitor of thymocyte proliferation.

The anterior chamber of the eye is an immunologically privileged site in which allografts survive longer than at other body sites. In this regard, it is relevant that aqueous humor (AH) inhibits lymphocyte proliferation. In order to analyze AH for specific substances that inhibit thymocyte proliferation, samples of human AH, murine AH, and rhesus monkey AH were added to cultures of thymocytes stimulated by IL-1 or IL-2 in the presence of PHA. All samples of AH tested had potent inhibitory activity on thymocyte proliferation in this system. Inhibitory activity was lost by heating AH to 80 degrees C for 1 h. Dialysis of murine AH indicated that species smaller than 3500 Da were capable of mediating this activity; we have termed the factor(s) responsible for this "small inhibitory factor(s) of AH." Retentate, containing species larger than 3500 Da, retained inhibitory activity, but less than nondialyzed AH. Assays for PGE2 demonstrated that murine and human AH contained small quantities of PGE2. These quantities were insufficient to inhibit thymocyte proliferation in our assay system. Furthermore, AH from mice treatend with indomethacin had full inhibitory activity. Assays for transforming growth factor beta (TGF-beta) after acid activation demonstrated significant quantities of latent TGF-beta within human and murine AH which could be largely neutralized by antisera to TGF-beta. Active TGF-beta "activity" was also present without acid activation in samples of AH at a level approximately 20% that of latent TGF-beta. However, most of this "activity" could not be neutralized by antisera to TGF-beta. AH contains factors capable of limiting thymocyte proliferation.

Immunologic evaluation of spontaneous regression of an intraocular murine melanoma.

The experiments reported here describe the derivation of an immunogenic melanoma cell line from B16 melanoma by sequential in vitro mutagenization with two chemical mutagens: n-methyl n-nitro n-nitrosoguanine (MNNG) and ethane methyl sulfonate (EMS). Following in vivo screening of over 100 mutant melanoma clones, a single clone was selected for further study. When transplanted to the anterior segment of the mouse eye, the mutant melanoma (D5.1G4) underwent spontaneous resolution in 20% of the immunologically intact hosts. Tumor rejection involved extensive necrosis and culminated in phthisis of the tumor-containing eye. Histologic analysis revealed a prominent mononuclear cellular infiltrate in contrast to the parental progressor B16 melanoma. Immunologic analysis of tumor-bearing hosts showed variable cytotoxic T lymphocyte (CTL) responses but potent delayed-type hypersensitivity (DTH) responses directed against the melanoma cells. Fluorescent activated cell sorter (FACS) analysis of tumor-infiltrating cells from ocular tumors revealed a cellular response consisting mainly of CD8+ CTLs and macrophages. Cultured D5.1G4 melanoma cells demonstrated: 1) enhanced expression of class I major histocompatibility complex (MHC) antigens; 2) increased susceptibility to CTL-mediated killing; and 3) increased susceptibility to tumor necrosis factor (TNF)-mediated cytolysis. Therefore, the intraocular D5.1G4 mutant melanoma model provides important insights into the immunology and immunopathology of intraocular tumor rejection. More intensive analysis of this intraocular melanoma may yield strategies for directing the immune response toward tumor rejection while minimizing damage to normal ocular components.

Emergence of a dominant cytotoxic T lymphocyte antitumor effector from tumor-infiltrating cells in the anterior chamber of the eye.

Previous studies in mice revealed that resolving intraocular tumors (UV5C25 fibrosarcoma) were infiltrated with mononuclear cells and invoked potent systemic delayed-type hypersensitivity responses without nonspecific tissue destruction. The present study characterized the tumor-infiltrating lymphocyte (TIL) population and established its role as the mediator of specific intraocular tumor rejection. This was accomplished by (a) isolating TIL from resolving intraocular tumors; (b) identifying characteristic surface markers on TIL; and (c) demonstrating in vitro and in vivo antitumor functions. Fluorescence-activated cell sorter analysis of TIL showed 33.4% Thyl+, 19.8% CD8+, 11.1% CD4+, 17.2% MAC-1+, 10.4% F4/80+, and 7.7% B220+. Functional studies indicated that TIL were directly cytolytic for UV5C25 tumor cells. Additionally a tumor-necrosis-factor(TNF)-sensitive cell line (WEHI 164.1) was lysed on cocultivation with TIL, whereas UV5C25 tumor cells were insensitive to lysis by TNF. Precursor CTL analysis demonstrated a high frequency (1/251) of tumor-specific precursors and a low frequency of alloresponsive cells in the TIL population. In vivo analysis by a Winn-type assay demonstrated that only TIL could effect tumor resolution in immunosuppressed hosts. These results demonstrate that although CD4+ T cells and macrophages were present and TNF activity was detected in the TIL population, there was no evidence for nonspecific tissue destruction within the eye. Therefore, this pattern of intraocular tumor rejection is mediated by a lymphocyte population expressing cell-surface phenotypes and functional characteristics of conventional cytotoxic T lymphocytes. Moreover, the results suggest that a regulatory mechanism within the eye allows for the emergence of one dominant antitumor effector (CTL) while controlling a more destructive mechanism (delayed-type hypersensitivity).

Immunological analysis of a destructive pattern of intraocular tumor resolution.

Spontaneous rejection of syngeneic intraocular P91 mastocytoma occurred by a T cell-dependent immune process that produced extensive, irreparable damage to normal ocular tissue indicative of a delayed-type hypersensitivity (DTH) lesion. We investigated the contribution of other effector modalities in this tumor resolution. Antibody was not responsible for either tumor rejection of phthisis of the tumor-containing eye since passively transferred hyperimmune serum failed to produce either tumor rejection or the characteristic ocular lesions. Moreover, the destructive pattern of tumor rejection occurred in splenectomized hosts which had depressed anti-P91 serum antibody and in a congenic strain (B10.D2oSn) lacking the C5 complement component. The likelihood that tumor necrosis factor (TNF) was involved in this pathogenesis was also ruled out by the observation that P91 tumor cells were not susceptible to lysis by recombinant TNF. A different pattern of tumor resolution was observed when the opposite eye was challenged with tumor. In this case, the tumor was rejected without damaging any major ocular structures. Histological analysis demonstrated a mononuclear cellular infiltrate which was identified by immunohistology to be Thy 1+, Lyt 2+. Thus, although both forms of cell-mediated immunity are available to the host, only one predominated in each eye. These results suggest that unique immunological control mechanisms influence the outcome of tumor resolution in the eye.

Destructive and nondestructive patterns of immune rejection of syngeneic intraocular tumors.

Two immunogenic, syngeneic murine tumors were used to analyze the immunopathological processes associated with the immune rejection of primary intraocular tumors. Intracameral inoculation of P91 mastocytoma, an immunogenic variant of P815 mastocytoma, into DBA/2 mice resulted in progressive tumor growth for several weeks before immune rejection eradicated the intraocular neoplasm. The histopathologic characteristics of the tumor rejection included: a) destruction of the vascular endothelium of the microvasculature feeding the tumor; b) ischemic bulk necrosis; c) extensive innocent bystander damage to normal ocular structures; and d) absence of direct inflammatory cell-to-tumor cell contact. Thus, the immunopathological features resembled a delayed-type hypersensitivity (DTH) lesion. A second intraocular tumor model was similarly studied. UV5C25 fibrosarcoma grew slowly in the eyes of syngeneic BALB/c hosts. In sharp contrast to P91 tumors, a mononuclear cellular infiltrate was prominent within the tumor. After 5 wk, the intraocular tumors were completely rejected without detectable damage to normal ocular structures. The rejection of UV5C25 tumors did not produce scar tissue, damage to vascular endothelium, bulk necrosis, or atrophy of the globe. Although tumor-specific cytotoxic T lymphocytes (CTL) and DTH responses were readily detected, there was no histological evidence for DTH-mediated tumor rejection. Moreover, in situ immunoperoxidase staining indicated that the majority of the infiltrating lymphocytes were CTL, based on their characteristic phenotype: Thy-1+, Lyt-2+. Furthermore, the growth of UV5C25 fibrosarcoma in athymic, natural killer (NK) cell competent BALB/c nude mice demonstrated progressive tumor growth without infiltrating host cells. Collectively, the results indicate that immunogenic intraocular tumors can undergo strikingly different patterns of immune rejection with profoundly different pathological consequences. In one case (P91), tumor rejection occurs by a process that strongly resembles DTH and produces extensive nonspecific damage to normal tissues, resulting in irrevocable loss of vision. In contrast, the second intraocular tumor undergoes an immune rejection that is characterized by precision and a notable absence of damage to normal ocular tissues. The weight of evidence presented here strongly supports the hypothesis that the latter form of tumor rejection is mediated by CTL. Thus, the immunologic pathway invoked for tumor rejection in the eye has a profound effect on the fate of this delicate organ and the preservation of vision.

Immune rejection of metastases arising from intraocular tumors in mice.

The role of the immune response in the elimination of spontaneous metastases arising from intraocular tumors was examined in a syngeneic intraocular murine tumor model. P91 mastocytoma (DBA/2 origin) expresses strong tumor-specific transplantation antigens and grows transiently in the eyes of syngeneic hosts before undergoing spontaneous rejection. An organ culture technique was used to detect spontaneous metastases in the lungs, spleens, brains, and thymuses of intraocular tumor-bearing mice. Metastatic tumor cells were detected in all organs of immunodeficient mice (i.e., athymic, nude, or x-irradiated DBA/2 mice) within 14 days of intraocular transplantation, and grew progressively thereafter. By contrast, metastatic tumors were rejected in 100% of the immunocompetent DBA/2 mice examined on day 15. Timed enucleation experiments demonstrated that the immune rejection of disseminated tumor cells occurred within 24-48 hr of their arrival at the various organs. The immune rejection of spontaneous metastases could be adoptively transferred to immunodeficient tumor-bearing mice using spleen cells, but not immune serum, from intraocular tumor-bearing immunocompetent donors. Selective cell depletion experiments revealed that the immune spleen cell effecting immunity was an Lyt 1+, 2+ T cell. The results indicate that the immune rejection of the spontaneous metastases arising from primary intraocular tumors is a T cell-dependent, radiosensitive process that rapidly eliminates metastases within the lungs, brain, thymus, and the spleen of the immunocompetent host.