Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

Reactive lymphocytes in lacrimal gland and vasculitic renal lesions of autoimmune MRL/lpr mice express L3T4.

The lacrimal gland inflammatory lesions and renal vasculitic lesions of autoimmune MRL/lpr mice were analyzed for the lymphocyte subsets present. The majority of cells were Thy-1.2+ T cells (mean, 85%) of the L3T4+ helper T phenotype (mean, 64 and 58%, respectively). Lesser numbers of Lyt-2+ suppressor/cytotoxic T cells, B cells, and macrophages were present. The finding that the majority of lymphocytes in both the lacrimal gland inflammatory lesions and renal vasculitis of MRL/lpr mice expressed L3T4 suggests that these cells may be capable of responding to antigen presentation and that an active immunologic response occurs at these sites.

The effect of sea star coelomocyte extract on cell-mediated resistance to Listeria monocytogenes in mice.

Mice treated with sea star factor (SSF), a protein extracted from sea star coelomocytes, became highly susceptible to infection with a normally sublethal dose of Listeria monocytogenes. This was in contrast to the expected result of increased resistance originally postulated because of the macrophage-activating properties of SSF. Enhanced susceptibility was seen when SSF was given from 96 h before to 48 h after infection with Listeria. Mice pretreated with SSF failed to develop immunity to Listeria when given a dose of organisms capable of immunizing nontreated mice. Treatment of immune mice with SSF did not alter their immune status. In addition, incubation of immune lymphocytes with SSF in vitro did not alter their ability to adoptively transfer immunity to normal recipients. Immune lymphocytes treated with SSF and then incubated with anti-SSF and C did, however, lose the ability to transfer immunity. These results suggest that SSF enhances infection by binding to T lymphocytes, inhibiting their replication upon contact with Listeria antigen and thus preventing the generation of a population of sensitized lymphocytes capable of effecting anti-Listeria immunity.

Recombination of heavy and light chains of human gamma-a-myeloma proteins: formation of hybrid molecules and configurational specificity.

The present studies demonstrate that the conditions necessary for reductive cleavage, isolation, and recombination of L and H polypeptide chains of human gammaA-myeloma globulins parallel those required for similar manipulation of the component chains of gammaG-globulin. Specificity of recombination was shown for chains derived from the same protein. In contrast, no intradass preferential recombination was demonstrable. Hybrid molecules, formed by reassociation of noncovalent bonds, could be synthesized from isolated chains of two immunoglobulin classes resulting in the formation of molecules of the type gammaA-H-gammaG-L and gammaG-H-gammaA-L. Several sera containing both gammaA- and gammaG-"monoclonal" peaks were studied, one of which demonstrated the L chains associated with both peaks to be identical both by electrophoretic mobility in acid-urea gel and antigenic analysis. The possibility is considered that this case represents a naturally occurring analogue of the artificially produced hybrid molecules described in this study. Configurational antigenic specificity of gammaA-myeloma proteins, imposed by the presence of kappa L chains in native and appropriately recombined molecules, provides a further indication of the importance of noncovalent bonds in the establishment of the quaternary structure of these proteins.

Nonspecific signals for B-cell localization and activation.

Virgin, inactive mammary gland autografted to the anterior chamber of the rabbit eye remains free of lymphoid cells. Activation of the ectopic gland by systemic injection of chorionic gonadotropin results in maturation of the gland and milk production, accompanied by the immirgration of lymphocytes and their activati-n to Ig formation, predominantly of the IgA class. In the presence of antigen-induced intraocular inflammation, the activated gland is able to influence the Ig class of B cells in the neighboring ocular tissues. These data suggest that even nonlymphoid tissues may elaborate lymphocyte-homing and polyclonal B-cell activating factors which function independently of specific antigen.

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation.

Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate.

Intraocular tumor suppression of retinoblastoma gene-reconstituted retinoblastoma cells.

Human retinoblastoma is caused by mutational inactivation of the retinoblastoma suppressor gene (RB). We have examined intraocular tumorigenicity of retinoblastoma cells in which RB expression was achieved by retroviral transduction. Retinoblastoma cells were injected into the anterior chambers of severe combined immunodeficient mouse eyes, and tumorigenicity was assessed. RB-expressing retinoblastoma cells usually failed to form progressive tumors in the anterior chamber, whereas the parental, RB-negative line, WERI-Rb27, was rapidly tumorigenic. These results support the hypothesis that inactivation of the RB gene is critical for the growth of retinoblastoma tumors. The potential use of RB reconstitution for treating human retinoblastoma is suggested by our finding that intraocular tumor growth can be suppressed by RB expression.

Ocular inflammation in MRL/Mp-lpr/lpr mice.

A systemic autoimmune disease spontaneously developed in MRL/Mp-lpr/lpr (MRL/lpr) mice. The disease was characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. Among the many autoimmune lesions are focal ocular inflammatory infiltrates involving the choroid and sclera. Of 104 mice examined histologically, the sclera was most often involved, with 30% of mice 5 months of age or older having scleral lesions that were often centered around small arteries. The choroid was the second most frequently involved tissue, with focal inflammation developing in 16% of these mice. Immunohistologic analysis of the ocular infiltrates showed that most of the mononuclear cells seen were L3T4 + T cells (CD4+ helper T cells), suggesting that the process was largely T cell-mediated. Smaller numbers of B cells and Lyt 2+ T suppressor/cytotoxic cells were seen. No such lesions were seen in congenic MRL/Mp- +/+, (NZBxNZW) F1 hybrid mice, and control BALB/c mice; vasculitis did not develop in all of the mice. These results suggest that the ocular lesions in MRL/lpr mice may be a model for ocular involvement in patients who have systemic necrotizing vasculitis.

Attempted oral immunization with chlamydial lipopolysaccharide subunit vaccine.

The effects of oral immunization with a recombinant vaccine expressing chlamydial lipopolysaccharide (LPS) on subsequent ocular challenge with Chlamydia trachomatis were studied in cynomolgus monkeys. Groups of four or five monkeys were given an oral vaccine containing 5 X 10(8) parent or recombinant Escherichia coli on days 0, 14, and 35 and were challenged with either 2 X 10(3) or 5 X 10(3) inclusion forming units of viable purified elementary bodies on day 42. On clinical and microbiologic grounds, oral immunization failed to protect monkeys against subsequent ocular challenge. Antichlamydial IgG or IgA antibodies were not induced by oral vaccination, and the antibody response following ocular challenge was similar in vaccinated and nonvaccinated animals. Paradoxically, however, while nonvaccinated control animals developed antibodies against chlamydial LPS detectable by immunoblotting after chlamydial challenge, the LPS vaccinated animals did not. This study demonstrates that the oral recombinant vaccine expressing chlamydial LPS was ineffective in protecting against chlamydial eye infection and strongly suggests that chlamydial LPS may not be an important antigen for protective immunity against chlamydia.

Murine models of Sjögren's syndrome. Immunohistologic analysis of different strains.

Lacrimal gland inflammation develops in several strains of autoimmune mice, including MRL/Mp-lpr/lpr (MRL/lpr), MRL/Mp-+/+ (MRL/+), and NZBxNZW F1 hybrids (NZB/W). These mice all develop an autoimmune disease characterized by glomerulonephritis and autoantibody formation, but each strain has unique clinical features and immunologic abnormalities. Previous studies have suggested that the intrinsic immunologic defect in MRL/lpr mice may be at the level of T cells, while in NZB/W mice it appears to be B cell-mediated. Immunohistologic analysis of the lacrimal gland lesions was performed on all three strains. Although T cells predominated (MRL/lpr 85%, MLR/+ 78%, and NZB/W 57%), differences in the immunohistologic profiles did exist. NZB/W mice had a significantly higher percentage of B cells (33% vs. 10% for MRL/lpr and 13% for MRL/+) and a correspondingly lower percentage of T cells. MRL/lpr mice differed from MRL/+ mice in that they exhibited a significantly higher percentage of helper T cells (63% vs. 49%) and a lower percentage of suppressor/cytotoxic T cells (14% vs. 30%). Class II antigen expression could be detected on the mononuclear cells at inflammatory sites within the lacrimal glands of all three strains, suggesting T cell activation and an active autoimmune immunologic event occurring in the lacrimal gland.

Autoimmune ocular disease in MRL/Mp-lpr/lpr mice is suppressed by anti-CD4 antibody.

MRL/Mp-lpr/lpr(MRL/lpr) mice spontaneously have a systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. Among the many autoimmune lesions present are focal ocular inflammatory infiltrates, involving the choroid and sclera. These lesions appear to be related to the vasculitis seen in MRL/lpr mice and are mediated by L3T4-positive helper T-cells (CD4-positive T-cells). Systemic treatment of MRL/lpr mice with a monoclonal anti-L3T4 antibody (anti-CD4) resulted in a dramatic reduction of both the frequency and severity of the ocular disease, supporting the hypothesis that the CD4-positive T-cells play an essential role in the pathogenesis of the choroiditis and scleritis in this strain.

Murine models of Sjögren's syndrome. Evolution of the lacrimal gland inflammatory lesions.

Lacrimal gland inflammation develops in a number of autoimmune mice, including the MRL/Mp-lpr/lpr (MRL/lpr), MRL/Mp(-)+/+ (MRL/+), and NZB x NZW F1 hybrid (NZB/W) strains. The authors studied the evolution of this process, MRL/lpr mice had inflammatory lesions at 4 weeks old. The lesions had enlarged by 2 months and were fully developed by 4 to 5 months of age. In MRL/+ mice, 4-week-old mice had no lesions, although some focal inflammation was detectable at 3 months old. Significant abnormalities were present at 6 months, and persisted and increased throughout life, with all mice having extensive lesions at 18 months or older. In NZB/W mice, the authors detected no lesions until 6 months of age, and these lesions were fully developed in 9 months. Immunocytochemical profiles, of the cell types infiltrating the lacrimal gland, showed differences not only between the strains, but also in each strain as inflammation progressed. All three types of mice had L3T4+ T cells as the major lymphocyte component, although MRL/+ had significantly more Lyt 2+ T cells than the other strains. NZB/W mice had significantly more B cells than the two MRL substrains. In both NZB/W and MRL/+ mice, there was a significant increase in the B cell population, and a decrease in the percentage of L3T4+ T cells. There was a significant decline in Lyt 2+ T suppressor/cytotoxic cells in both NZB/W and MRL/lpr mice. This last finding was consistent with the more rapid development of inflammation in these strains than in the MRL/+ mice, where Lyt 2+ T suppressor/cytotoxic cells persist. Together, these results indicate that the autoimmune response in murine models of Sjögren's syndrome is a dynamic, evolving process with strain-related changes in lymphocyte subsets.

Mechanisms of allograft rejection of corneal endothelium.

The local intraocular graft-vs.-host (GVH) reaction, involving the destruction of the corneal endothelial cells of the rabbit host by sensitized donor lymphoid cells, has been used to study the mechanism of corneal allograft rejection. Pretreatment of donor cells with a specific mouse monoclonal hybridoma anti-T cell antibody and complement suppresses the destructive reaction, suggesting that a cellular-immune mechanism is primarily involved. Pretreatment of donor cells with mitomycin-C completely abolishes the local GVH reaction, indicating that the effector lymphocytes must undergo mitosis within the eye before they can engage in target cell destruction. Finally, studies of the local GVH reaction in irradiated leukopenic recipients or in preinflamed rabbit eyes suggest that host leukocytes may contribute nonspecifically to enhance the destructive process. These studies show that the local ocular GVH reaction may provide a useful model for the study of the mechanisms involved in the rejection of corneal allografts.

Immunology of corneal allograft rejection: in vitro sensitization of effector cells.

Anterior chamber injection of donor rabbit lymphocytes sensitized in vitro to recipient alloantigens leads to the development of small focal areas of endothelial cell destruction (pocks) on the recipient cornea. Damage may be observed through a specular microscope as early as 2 days after injection of sensitized lymphocytes. Recipients of unsensitized allogeneic or sensitized autologous lymphocytes demonstrate little or no endothelial damage and no pock formation. Flat endothelial preparations reveal focal destruction of the endothelium with multiple foci, many infiltrated and surrounded by mononuclear cells. This model provides controlled sensitization to a variety of histocompatibility and corneal antigens that may be responsible for initiation of graft rejection.

Immunology of corneal allograft rejection: HLA-DR antigens on human corneal cells.

Human stromal and endothelial cultures were established from corneas obtained from the Maryland Eye Bank. Cultures were incubated in the presence of human gamma interferon for 4 days, trypsinized, washed, and then stained with a monoclonal reagent specific for human Class II (HLA-DR) antigens. Under these conditions, 100% of human corneal endothelial cells and 40 to 70% human corneal stromal cells expressed HLA-DR antigens.

A role for T lymphocytes in preventing experimental herpes simplex virus type 1-induced retinitis.

We have previously shown that herpes simplex virus Type 1 (HSV-1) inoculated into the anterior chamber (AC) of one eye of BALB/c mice results in retinal destruction in the opposite eye while retinas in virus-injected eyes are preserved. In the present study, immunodeficient mice (athymic BALB/c or normal BALB/c which had received either gamma-irradiation [450 R] or cyclophosphamide [150 mg/kg treatment]), demonstrated bilateral retinal destruction upon unilateral AC inoculation of HSV-1. Reconstitution of these immunodeficient mice with spleen cells obtained from days 10-21 AC-inoculated donor mice, prior to AC inoculation of HSV-1, prevented retinal necrosis in more than 80% of both eyes. In contrast, donor cells from mice inoculated subcutaneously (SC) with HSV-1 preserved only about 30% of recipient retinas, regardless of the cell transfer time after donors received HSV-1. Normal unsensitized syngeneic donor spleen cells failed to prevent bilateral retinal necrosis in either athymic or irradiated BALB/c mice, although they eliminated recipient mortality. T cell depletion of AC- or SC-inoculated donor cells removed their retinal protective effects completely. These studies demonstrate an active role for T lymphocytes in controlling the extent of disease in a murine model of HSV-induced retinitis.

The role of Fas-Fas ligand-mediated apoptosis in autoimmune lacrimal gland disease in MRL/MpJ mice.

PURPOSE: MRL/MpJ mice spontaneously develop lacrimal gland inflammation and are a model for the human disorder Sjögren's syndrome. MRL/MpJ-lpr/lpr (MRL/lpr) and MRL/Mp-+/+ (MRL/+) mice are congenic substrains, which differ only by a single autosomal recessive gene, the lpr mutation. This mutation results in defective Fas protein, defective lymphocytic apoptosis, and accelerated autoimmune lacrimal gland disease in MRL/lpr mice. We evaluated apoptosis in the lacrimal glands of MRL/lpr and MRL/+ mice. METHODS: Inflammatory cells in the lacrimal glands of MRL/lpr and MRL/+ mice were evaluated for apoptosis with TUNEL staining and Fas and Fas ligand expression with immunohistochemistry. RESULTS: MRL/lpr mice had a greater percentage of the lacrimal gland replaced by inflammatory infiltrate (30.3% +/- 7.0%) than did MRL/+ mice (13.0% +/- 3.0%, P = 0.02). However, similar amounts of lymphocytic apoptosis were present in the lacrimal glands of MRL/lpr and MRL/+ mice. The mean number of apoptotic cells per unit area of inflammation was 23.8 +/- 2.4 in MRL/lpr mice and 24.6 +/- 6.0 in MRL/+ mice (P = 0.91). Fas expression was absent on lymphocytes in MRL/lpr mice but was present on lymphocytes in MRL/+ mice. Fas ligand expression was present on epithelial structures in both substrains. CONCLUSIONS: The accelerated lacrimal gland disease inflammation in MRL/lpr mice does not appear to be due to decreased apoptosis in the microenvironment of the lacrimal gland of MRL/lpr mice. It appears that in MRL/lpr mice there is defective extrathymic lymphoid apoptosis, permitting a relatively greater expansion of autoreactive T cells, which subsequently invade the lacrimal gland.

Paradoxic effect of anti-CD4 therapy on lacrimal gland disease in MRL/Mp-lpr/lpr mice.

PURPOSE: MRL/Mp-lpr/lpr mice (MRL/lpr) spontaneously develop lacrimal gland inflammatory lesions and are a model for the human disease Sjögren's syndrome. Therapy with monoclonal antibodies (mAb) to CD4 ameliorates the autoimmune renal, vasculitic, and intraocular inflammatory lesions in MRL/lpr mice. The effect of anti-CD4 mAb therapy on lacrimal gland immunopathology was evaluated. METHODS: From 1 to 5 months of age, MRL/lpr mice were treated with weekly intraperitoneal injections of 2 mg anti-CD4 mAb, after which they were killed and their lacrimal glands were removed for histologic evaluation and immunocytochemistry. Control mice were administered weekly intraperitoneal injections of either saline or normal rat immunoglobulin. RESULTS: Anti-CD4 mAb treatment produced no reduction in lacrimal gland inflammation but did change its morphology. In control mice, there were multiple sharply delineated foci of inflammatory cells in the lacrimal gland, whereas in anti-CD4 mAb-treated mice, there was a more diffuse inflammation surrounding ill-defined foci that spread throughout the gland. Immunocytochemistry revealed that in control mice, lesions were composed predominantly of CD4+ T cells, but in anti-CD4 mAb-treated mice, CD8+ T cells predominated. CONCLUSIONS: Although anti-CD4 mAb therapy of MRL/lpr mice eliminated autoimmune renal disease, autoantibody formation, and ocular inflammatory disease, it had a paradoxic effect on lacrimal gland lesions. Lacrimal gland lesions in the anti-CD4 mAb-treated mice were not decreased, but they had a different morphology and a different immunocytochemical profile.

Experimental autoimmune dacryoadenitis. I. Lacrimal gland disease in the rat.

Experimental autoimmune dacryoadenitis was produced in Lewis rats by immunization with a single intradermal administration of a 3M KCl extract of exorbital lacrimal gland in CFA, when enhanced by simultaneous i.v. injection of killed Bordetella pertussis. No significant lacrimal lesions were observed in control animals immunized with the extracts of Harderian or salivary glands. Gel filtration of the 3M KCl extract on Sephacryl S-300 column yielded three protein fractions. Only fraction III (MW = 10-55K) induced marked dacryoadenitis following a single injection of 2.0 mg protein in CFA plus pertussis. The infiltrates in the exorbital lacrimal lesions were first apparent around the ducts and associated vasculature. From this area, the infiltrates appeared to spread to the acini drained by these ducts, ultimately involving as much as 30-50% of the gland. The affected glands most commonly showed a diffuse nongranulomatous infiltrate of small lymphocytes, macrophages, and plasma cells; this was focal in nature, involving acinar atrophy and breakdown, and replaced the normal architecture in extreme cases. The Harderian and salivary glands were uninvolved in these animals, suggesting a restricted specificity of this response. Lewis rats immunized with exorbital lacrimal gland fractions I or II in CFA plus pertussis showed only minimal lesions, similar to controls receiving CFA and pertussis without antigen. These findings suggest that an autoantigen exists in the lacrimal gland of the rat that is capable of inducing a specific lymphoproliferative dacryoadenitis.