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BACKGROUND: Although many lines of evidence suggest an autoimmune etiology, the pathophysiology of opsoclonus-myoclonus syndrome (OMS) remains poorly understood and no immunologic abnormalities have correlated with neurologic severity. Conventional immunotherapies often do not prevent relapse or permanent sequelae. OBJECTIVE: To test the cellular immune hypothesis of OMS in a cross-sectional study and determine if CSF lymphocyte subset analysis provides biomarkers of disease activity. METHODS: The expression of lymphocyte surface antigens was investigated in CSF and blood of 36 children with OMS and 18 control subjects, using a comprehensive panel of monoclonal antibodies to adhesion and activation proteins in combination with anti-CD3 and anti-CD45 antibodies in four-color fluorescence-activated cell sorting. RESULTS: Although most children with OMS had normal CSF cell counts, they exhibited expansion of CD19+ B-cell (up to 29%) and gammadelta T-cell (up to 26%) subsets and a lower percentage of CD4+ T-cells and CD4/CD8 ratio, which persisted even years after disease onset and conventional treatments. The percentage of activated CSF T-cells was also higher. Abnormalities correlated with neurologic severity, as scored blinded from videotapes using a 12-item motor scale, and disease duration. No significant differences were found between tumor and no-tumor groups. In children with neuroblastoma, tumor resection or cancer chemotherapy did not alter immunologic abnormalities. CONCLUSIONS: CSF B- and T-cell recruitment is linked to neurologic signs in pediatric OMS, which may relate to relapses and disease progression.

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The state of pregnancy is an immunological enigma during which the body must prevent rejection of the antigenically foreign fetus while at the same time maintain sufficient maternal host defense mechanisms to combat infection. Although most studies on the immunology of pregnancy focus on immune suppression, several studies have shown an increase in nonspecific host defense, which is postulated to be a compensatory mechanism for decreased specific immunity during pregnancy. Studies in this laboratory have shown that monocyte surface FcgammaRI (CD64) and FcgammaRII (CD32) expression progressively increase throughout pregnancy, while surface MHC class II expression remains unchanged. Functional studies revealed that the number of phagocytic monocytes which could be isolated from pregnant women was increased. These cells exhibited an increased capacity to ingest IgG-opsonized human erythrocytes. This study shows for the first time that monocyte surface FcgammaR expression and FcgammaR-mediated functions are increased during pregnancy. These results support the hypothesis that nonspecific immunity as represented by FcgammaR expression and function is increased during pregnancy.

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CTLL-2 cells are a clone of CTL that are dependent on IL-2 for proliferation. In addition to various cytokine receptors, we observed that these cells express three subtypes of adenosine receptors (ARs). In an initial attempt to delineate the functions of these receptors in CTLL-2 cells, we tested their role in proliferation. Elimination of endogenous adenosine with adenosine deaminase (ADA) markedly suppressed IL-2-dependent proliferation of these cells. This proliferative response was restored by addition of R-phenylisopropyladenosine (R-PIA), a non-hydrolyzable adenosine analogue. The stimulatory response to R-PIA was attenuated following blockade of ARs by 0.5 mM theophylline and 10 microM BW-A1433, but not by blockade of the A1AR with 100 nM xanthine amine congener. The rank order of potency of adenosine analogues in proliferation assays was R-PIA > or = N-ethylcarboxamide adenosine > S-PIA > PAPA-APEC (a substituted ethylamino-5'-N-ethylcarboxamidoadenosine). These data suggest a potential role of the A3AR in the proliferative response. R-PIA stimulates production of 1,4,5-inositol trisphosphate in CTLL-2 cells, suggesting a role of the phospholipase C signaling pathway in the proliferative response. A23187 (100 nM) and phorbol 12,13 dibutyrate (10 nM), but not 4 alpha-phorbol (10 nM), were able to restore IL-2-dependent CTLL-2 proliferation in the presence of ADA. Furthermore, inhibition of protein kinase C by staurosporine (10 nM) and of phospholipase C by tricyclodecan-9-yl-xanthogenate (D609) blocked R-PIA-mediated cell proliferation. These data demonstrate an obligatory role of adenosine in IL-2-dependent proliferation of CTLL-2 cells and support the involvement of an AR-stimulated phospholipase C signaling pathway in this process.

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Injection of an antigen into the anterior chamber induces an immune response in which antibody production is normal while delayed hypersensitivity reactivity is depressed. Several antigens have been used to induce this response which has been termed anterior chamber associated immune deviation. We have demonstrated that allogeneic lymphocytes injected into the anterior chamber of Lewis rats increase the success rate of subsequent corneal grafts derived from the lymphocyte donor strain. To begin understanding the antigenic requirements of this phenomenon, Wistar/Furth lymphocytes were partially purified into B- and T-cell populations by panning on anti-immunoglobulin coated petri-dishes. These enriched populations were injected separately into the anterior chamber of Lewis rats. Two weeks later these Lewis rats received a full-thickness corneal graft derived from Wistar/Furth donors. Grafts were scored for opacity and neovascularization over the subsequent 8-10 weeks. In control animals injected with balanced salt solution, 20% of the grafts cleared sufficiently to be judged successful. Grafts placed on rats injected with unseparated splenic lymphocytes were judged successful in 75% of the cases. Comparable percentages for grafts on animals injected with B-cell enriched and T-cell enriched populations were 85 and 50 respectively. These results suggest that B cells, which express both class I and class II major histocompatibility antigens are more efficient at inducing anterior chamber associated immune deviation than are T cells, the majority of which express only class I major histocompatibility antigens.

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Rhodopsin, a membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) in Lewis rats. Synthetic peptides derived from rhodopsin sequences that cover hydrophilic, exposed regions of the protein were tested for their capacity of eliciting in vitro T cell proliferation and their ability for inducing EAU in Lewis rats. Rats were injected with rhodopsin's peptides mixed in complete Freund's adjuvant containing M. tuberculosis H37Ra (5 mg/ml) three days after pretreatment with cyclophosphamide (20 mg/kg). ELISA results indicate that all peptides induce antibody responses; however antibody titers differ among sera tested. Immunization with four peptides--the amino-terminus (2-32), loop I-II (61-75), loop V-VI (230-251), and the carboxyl-terminus (324-348 and 331-342) induced both antibody and T cell responses. In all cases, the proliferative responses of cells derived from peptide-injected rats were stronger against the immunizing peptide than against native protein. Three distinct uveitogenic epitopes were identified on rhodopsin's cytoplasmic surface--within the rhodopsin carboxyl-terminus (324-348), loop I-II (61-75), and loop V-VI (230-250). Histopathologically, at the immunized doses, total destruction of the photoreceptor cell layer was observed as compared to the control group. Loop V-VI caused severe inflammation of the retina while the other pathogenic peptides produced less severe destruction with few inflammatory cells present. Our study indicates that the major immunodominant T cell epitope (331-342) is also involved in EAU induction but is not the primary uveitogenic site.

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Rhodopsin, an integral membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) when injected into Lewis rats. This disease is characterized by a mononuclear and polymorphonuclear cellular infiltrate of the retina resulting in destruction of the photoreceptor cells. In this study the B and T cell specificities of the response to bovine rhodopsin by Lewis rats were determined. Antibodies induced by injection of rhodopsin were directed almost exclusively to the IV-V loop (residues 174-202). Later in the response, antibody to the N-terminus was also detected. At the T cell level, most activity was directed to the C-terminus as measured by in vitro lymphocyte proliferation. Other minor T cell epitopes were found in the II-III (96-114) and IV-V (174-202) loops. Further dissection of the amino acid sequence responsible for the activity isolated to the C-terminus indicated that a 12-amino acid-long sequence (331-342) elicited the strongest proliferative response.

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In an attempt to overcome the lack of well characterized, inbred, and congenic strains, as well as a paucity of immunological reagents, in rabbit and rat models, we have developed a procedure for performing full-thickness, penetrating keratoplasty in the mouse. One hundred percent of isografts (BALB/c onto BALB/c) were successful, while 83.3% of allografts (C3H onto BALB/c) failed. The use of a mouse model to study corneal graft failure will allow a critical evaluation of the role of a variety of immunological mediators in causing graft failure.

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Failure of corneal grafts is thought to involve the development and activation of specifically sensitized T-cells. One method which might be used to circumvent the development of such cells is the phenomenon of anterior chamber-associated immune deviation (ACAID). Injection of antigen into the anterior chamber of the eye leads to an immune response characterized by normal antibody response coupled with depressed T-cell reactivity especially as measured by delayed-type hypersensitivity. To determine if this phenomenon could be used to alter the course of graft failure, potential recipients (Lewis rats) were injected intracamerally (IC) with allogeneic lymphoid cells (Wistar-Furth). Orthotopic, full-thickness, penetrating keratoplasty was done 0-30 days later, and the recipients were observed for at least 60 days. Approximately 75% of Wistar-Furth corneal grafts placed on uninjected Lewis rats failed as evidenced by continued opacity, edema, and infiltration of mononuclear cells into the grafts. The IC injection of Wistar-Furth lymphocytes decreased this failure rate to 25% and 50% when grafting was done 14 and 7 days after injection, respectively. Grafts of cornea from a third strain onto IC injected animals failed at an intermediate rate which demonstrated some immunologic protection. The results of these studies indicate that IC injection of allogeneic lymphocytes results in prolonged acceptance of corneal grafts syngeneic with the injected lymphocytes.

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The fate of orthotopic, allogeneic corneal grafts was followed in inbred rats following three different grafting procedures. Corneas were grafted to recipient rats either in an intralamellar pocket, on a deep stromal bed or by penetrating keratoplasty. In all cases the donor cornea consisted of all three tissue layers (epithelium, stromal and endothelium). Recipient corneas were pretreated with lipopolysaccharide to induce neovascularization. In all three experimental groups, allogeneic grafts went through a phase which clinically resembled a rejection. This phase was characterized by the development of vessels into the transplanted cornea, and opacification. This phase peaked at between 2 and 3 weeks after which some of the grafts cleared and the vascularization receded. Approximately 65% of grafts placed on a deep stroma bed cleared and were considered successful while only 30-35% of the grafts placed in intralamellar pockets or in full-thickness defects were deemed successful. Histological observations paralleled these clinical impressions.

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Intralamellar keratoplasty in inbred rats is a valuable method for the investigation of corneal allograft rejection. Present methods of intralamellar corneal grafting are tedious and time-consuming. In addition, induction of neovascularization, a prerequisite to inducing graft rejection, is variable. In an effort to overcome these problems, the authors have developed an improved method of intralamellar keratoplasty. Intralamellar pockets are formed by introducing a 30-g needle into the corneal stroma near the limbus. Approximately 10-25 microliters of lipopolysaccharide (LPS) (100 micrograms/ml) is injected into the stroma, forming a stromal bleb. This bleb is incised to form a pocket, resulting in the leakage of the injected liquid. The intralamellar pocket formation is completed by dissecting any remaining stromal fibers in the area of former bleb. Once the pocket has been formed, grafts of corneal tissue are inserted, and the incision is closed. This method of keratoplasty has the following advantages over previously reported methods: (1) It is more rapid and less tedious because the formation of the corneal bleb protects the anterior chamber from being invaded during the corneal incision; (2) It leads to a reproducible induction of neovascularization in every cornea so treated; (3) It results in a higher frequency of allograft rejection.

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Spleen cells from normal, nonautoimmune mice (C3H/HeN) spontaneously produce hemolytic plaques against autologous bromelain-treated red blood cells (BrMRBC). The number of anti-BrMRBC plaques detectable can be increased by including either a 3 M KCl extracted antigen from BrMRBCs or the hapten phosphorylcholine chloride (PC) as an antigenic analog in the plaque assay. Optimal PC concentration for augmenting the number of plaque forming cells (PFCs) was between 10(-7) and 10(-8) M. Incubation of spleen cells with an equal volume of 10(-7) M PC one, two, or three times resulted in the preparation of populations of cells which yielded increased numbers of PFCs. In addition, the number of anti-BrMRBC plaques of cells incubated three times could not be further increased by adding PC to the plaquing mixture. The eluate produced by the initial incubation of spleen cells with 10(-7) M PC specifically suppressed the anti-BrMRBC PFC response of these nonhapten augmentable cell populations (3 X eluted). These studies indicate that a naturally occurring autoantibody response is normally regulated by the presence of a molecule bound to the cell surface of autoantibody forming cells.

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Low level irradiation (400-500 R) of normal mice or of murine spleen cells resulted in the detection of an enhanced number of plaque-forming cells against bromelain-treated autologous red cells (Br MRBC) 1 day later. The mechanism responsible for the increased numbers of plaques is apparently the elimination of a suppressor T cell since the addition of thymocytes or of Lyt 1+2+ splenic cells to cultures of irradiated cells reversed the radiation-induced increase. Studies on the ontogeny of the phenomenon indicate that anti-Br MRBC plaques can be formed by spleen cells taken from mice shortly after birth although adult levels are not reached until after 3 weeks of age. Radiation-induced increases in the number of plaques were not seen until 3 weeks of age, thus, suggesting a temporal developmental sequence of the ability to produce autoantibodies and to regulate such production.

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Normal mice possess spleen cells capable of forming hemolytic plaques against bromelain-treated autologous red cells (Br MRBC). There is present in the serum of these same mice a substance which can inhibit the formation of these plaques. This substance is inhibitory to the secretion of these antibodies following incubation of spleen cells in 20% serum at 4 degrees C for 5 min. This substance is not inhibitory to the formation of anti-sheep erythrocyte plaques from mice either immunized or nonimmunized with sheep erythrocytes. Characterization of the substance indicates that it is neither soluble antigen nor specific antibody. However, inclusion of nanogram amounts of soluble antigen from bromelain-treated red cells in the assay mixture effectively neutralized the suppression. In addition, passage of serum through a mouse anti-Br MRBC antibody immunoadsorbent effectively removed the suppressive activity of the serum while suppression could be recovered in the acid eluate from such a column. This suggests that the mechanism of suppression brought about by incubation in serum is due to the action of a molecule possessing anti-idiotypic activity directed against the cell surface receptors of anti-Br MRBC B cells. Attempts to isolate the molecule based on the postulate that it is immunoglobulin in nature have been unsuccessful.

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