Tropomyosin-induced arthritis in rats.

OBJECTIVE: Immunization of rats with alpha-tropomyosin (TPM) led to arthritis, uveitis and dermatitis, typical features of Behçet's disease (BD). The present study characterizes the arthritic features of this animal model, not previously described. METHODS: Lewis rats were immunized with bovine alpha-TPM and another group of rats was treated with neutralizing anti- tumor necrosis factor-alpha (TNF-alpha) antibodies. RESULTS: Clinically more than 90% of the immunized rats developed severe acute arthritis 12 days after vaccination. Rats that were followed-up for 6 months had persistent inflammation of the leg joints. Histologic studies demonstrated predominant mononuclear infiltrations in the acute phase of arthritis; the chronic arthritic process resulted in cartilage and bone damage and abundant fibrosis which led to joint deformations. Male and female rats had a similar clinical course. Analysis of the splenocyte cytokine profile kinetics revealed a persistently high level of interferon-gamma (INF-gamma) and an increase in TNF-alpha secretion during the acute phase. Increasing levels of interleukin (IL)-10 heralded the decline in clinical arthritis. No IL-4 was detected. No arthritis was detected in the rats treated with anti-TNF-alpha antibodies. CONCLUSION: The data indicates that alpha-TPM serves as an autoantigen to induce acute and chronic destructive arthritis in rats. This model is a TNF-alpha dependent autoimmune disease, with a Th1 cytokine profile.

A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity.

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.

Autoantibodies to tyrosinase: the bridge between melanoma and vitiligo.

BACKGROUND: Tyrosinase is an enzyme that participates in the process of melanin production in normal melanocytes and melanoma cells. Enzymes are known to be autoantigens in various autoimmune disorders; thus, after the detection of antityrosinase antibodies in patients with vitiligo and melanoma, tyrosinase was defined as an autoantigen in these conditions. In some patients with melanoma the disease is associated with the appearance of "vitiligo-like" white patches on the skin, called melanoma-associated hypopigmentation (MAH). In this article, the authors summarize the recent data related to antityrosinase antibodies and expand on their role in the pathogenesis of vitiligo, melanoma, and MAH. In addition, the beneficial clinical applications of antityrosinase antibodies are presented. METHODS: An enzyme-linked immunoadsorbent assay to detect the antityrosinase antibodies in the serum of patients and healthy volunteers was established using mushroom tyrosinase. Employing this method, antityrosinase antibodies were analyzed in a diverse group of patients with melanoma and vitiligo and in mice immunized with tyrosinase. RESULTS: In patients with melanoma, those with metastatic disease had a higher titer of antityrosinase antibodies compared with healthy subjects, whereas patients with MAH and those with no evidence of disease had similar titers to the control group. The titer of antityrosinase antibodies in patients with metastatic melanoma treated by vaccination with antiidiotypic antibodies mimicking the high molecular weight melanoma-associated antigen (HMW MAA) initially increased after the vaccination and then decreased. High titers of antityrosinase antibodies were detected in patients with diffuse vitiligo compared with patients with localized disease and with the healthy control group. Mice immunized with tyrosinase generated a high titer of antityrosinase antibodies and after the inoculation of melanoma cells developed a lower number of lung metastases compared with an unvaccinated control group. CONCLUSIONS: The appearance of antityrosinase autoantibodies in the serum of patients with metastatic melanoma and diffuse vitiligo is characterized by these two pathologies. The changes in the serum level of these autoantibodies in patients with melanoma after immunization with another antigen (HMW MAA) may have diagnostic and therapeutic implications.

Melanoma-associated hypopigmentation: where are the antibodies?

Antibodies to the B16 melanoma cell line and to tyrosinase have been recently defined in our laboratory in sera of patients with vitiligo, melanoma, melanoma-associated hypopigmentation (MAH), and in healthy subjects. The antibody titers in each subject were measured by enzyme-linked immunosorbent assay, were compared with the mean optical density (OD) of the control group, and were expressed as relative OD. The titers of anti-B16 antibodies (relative OD +/- standard error) were 1.000 (0.058) in the controls, 1.025 (0.077) in patients with metastatic melanoma, 0.5862 (0.15) in MAH, 1.377 in surgery-induced MAH, 1.087 in vaccination with anti-idiotypic antibodies, and 2.098 (0.15) in autoimmune vitiligo. The titers in vitiligo were significantly higher (p < 0.0001) than in MAH or in healthy controls. Antityrosinase antibodies were found in titers of 1.000 (0.1024) in the controls, 1.516 (0.225) in metastatic melanoma, 1.027 (0.180) in MAH, 1.075 in surgery-induced MAH, 2.308 in vaccination-induced MAH, and 4.536 in vitiligo. Differences were found between vitiligo and MAH (p = 0.008), surgery-induced MAH (p = 0.009), vaccination-induced MAH (p = 0.059), and healthy subjects (p < 0.0001). The results of this study point to the cross-antigenicity between melanocytes and melanoma cells, and to participation of antibodies against melanoma-associated membrane antigens in the mechanism leading to the development of MAH in patients with melanoma.

Superantigens and experimental SLE induced by idiotypic dysregulation.

OBJECTIVE: The effects of the superantigens (SAgs) Staphylococcal Enterotoxin B (SEB), Toxic Shock Syndrome Toxin-1 (TSST-1) and Mycoplasma Arthritidis Mitogen (MAM) were examined on the induction and on the course of experimental SLE-like disease. METHODS: Immunization of BALB/c mice with human anti-DNA mAb (MIV-7) carrying the pathogenic idiotype 16/6 emulsified in complete Freund's adjuvant (CFA), followed by a boost of MIV-7/PBS 3 weeks later, generated an experimental SLE via an idiotypic dysregulation. RESULTS: After immunization with MIV-7/SAg, replacing the MIV-7 boost by SAg, and then injecting SAg 7 weeks after the regular induction of the SLE-like disease, the mice failed to produce anti-hIgM and dsDNA Ab up to 6 months after the induction. The mice immunized with MIV-7/CFA and boosted with the SAg had high titers of anti-hIgM but no detectable anti-dsDNA Ab. In both experimental groups low titers of anti-CL Abs developed in 25/40 (62%) and 30/38 (79%) of the mice respectively, including the control mice immunized with non-pathogenic human IgM/SAg or PBS/SAg. The mice immunized according to the "classical" protocol showed increased titers of anti-dsDNA Ab (22%) and anti-CL Ab (28%) during 10 weeks of observation. In contrast SEB, TSST-1 and MAM induced a 29%, 1% and 17% reduction in the anti-DNA titers and a 32%, 15% and 12% reduction in the anti-CL titers, respectively. CONCLUSIONS: These data suggest that the SAg tested here cannot replace the effect of CFA in the induction of the primary humoral response. The SAgs TSST-1, SEB and MAM did not induce the SLE-like disease following idiotypic modulation. Moreover, they may have had a suppressive effect on the idiotypic network in our model. The appearance of anti-CL Abs in almost all the experimental groups including the naive mice supports the possibility that microbial SAgs can induce the production of autoantibodies by different mechanisms. The SAgs TSST-1, SEB and MAM reduced autoantibody production in the serologically established idiotypic-induced experimental SLE-like murine model. This beneficial effect may indicate new directions for research on the management of SLE.

Tyrosinase as an autoantigen in patients with vitiligo.

Vitiligo is considered an autoimmune disorder due to the generation and presence of autoantibodies directed against melanocyte antigens in the patients' sera. In the present study we point towards a newly defined autoantigen in vitiligo, the enzyme tyrosinase, which participates in the process of melanogenesis. Anti-tyrosinase antibodies were detected in the sera of seven patients with diffuse and 11 patients with localized vitiligo. Employing solid-phase ELISA to mushroom tyrosinase, we found that patients with diffuse vitiligo had significantly higher titres of IgG anti-tyrosinase autoantibodies than patients with localized disease or healthy subjects. These anti-tyrosinase autoantibodies have relatively high functional affinity to tyrosinase and can be recovered from vitiligo patients' sera by affinity purification. The anti-tyrosinase antibodies do not cross-react with other enzymes recognized as autoantigens in different autoimmune disorders and the autoantibodies do not block the enzymatic activity of tyrosinase, indicating that they are not reacting with the catalytic site of the enzyme. These data point to tyrosinase as an autoantigen in vitiligo and suggest that anti-tyrosinase titres can serve as a marker for disease activity.

Anti-tyrosinase antibodies in malignant melanoma.

Anti-tyrosinase antibodies were measured by enzyme-linked immunosorbent assay in sera of patients with malignant melanoma with either metastatic disease or no evidence of disease, in patients with melanoma and associated hypopigmentation (MAH), in patients with vitiligo and in healthy volunteers. The mean relative absorbance (Arel) was calculated by dividing the absorbance of each sample by the mean value for the control group. Using this method, the Arel of the control group was 1.000(SE 0.083). Arel of patients with metastatic disease (1.516; SE 0.225) was significantly higher (P = 0.03) than the value for the controls, but insignificantly higher than that for patients with no evidence of disease (1.216; SE 0.148). Patients with no evidence of disease, in whom the primary lesion originated in the lower limb, had a significantly higher (P = 0.01) Arel than the healthy volunteers. Patients with metastatic disease showed higher Arel if their primary lesions were confined to the area of the head and neck or to the lower limb. Patients with vitiligo had higher Arel values for their anti-tyrosinase antibody than any of the other groups. However, those with melanoma and MAH (vitiligo-like) had the same Arel of anti-tyrosinase antibodies as the controls or the patients with metastatic melanoma. This observation reflected the possible absorption of anti-tyrosinase antibodies to melanoma antigens, and pointed to the participation of anti-tyrosinase antibodies in the destruction of normal melanocytes in patients with melanoma, as part of the immune reaction towards this disease.

Reactivity to tyrosinase: expression in cancer (melanoma) and autoimmunity (vitiligo).

Anti-tyrosinase antibodies are found in the sera of patients with diffuse vitiligo, metastatic melanoma and in sera of patients with melanoma and hypopigmentation (MAH). The autoantigen is tyrosinase itself, the enzyme that participates in pigment (melanin) formation by both melanocytes and melanoma cells. The production of autoantibodies in both diseases is associated with the development of white patches on the patients' skin. The presence of these autoantibodies in patients with melanoma may suggest a better prognosis. Cross-antigenicity between melanoma cells and normal melanocytes is most probably the key mechanism leading to the appearance of MAH. Anti-tyrosinase antibodies are absorbed by melanocytes and by melanoma cells in all the 3 situations (melanoma, vitiligo, MAH). However, since the production of antibodies in vitiligo exceeds that in melanoma or MAH, the antibodies are detected in significantly higher levels only in vitiligo. It is suggested here that anti-tyrosinase antibodies may be responsible, or at least participate in destruction of normal melanocytes during the immune response to melanoma antigens. This mechanism may be responsible for the phenomenon of MAH in patients with melanoma, and for the formation of the autoimmune vitiligo. Anti-tyrosinase antibodies may serve for two clinical applications. One is a marker for monitoring and follow up of patients with melanoma treated by immune therapy. The second is active (or passive) immunotherapy. We have recently shown that C57BL/6J mice immunized with tyrosinase generated a high titer of antityrosinase antibodies, and following the inoculation of melanoma cells developed lower number of lung metastases, compared to the unvaccinated control group.

Anti-tyrosinase antibodies participate in the immune response to vaccination with anti-idiotypic antibodies mimicking the high-molecular-weight melanoma-associated antigen.

Seven patients with metastatic melanoma were vaccinated with anti-idiotypic monoclonal antibody (mAb) MK2-23 which mimics the high-molecular-weight melanoma-associated antigen (HMW MAA). Sera samples were assayed for anti-anti-idiotypic antibodies, by Ab1-Ab2 complex inhibition test, for anti-B16 epitope antibodies, which are a heterogeneous group against various antigens presented on B16 melanoma cells and for anti-tyrosinase antibodies, which are specific against tyrosinase. Our results pointed to the participation of anti-tyrosinase antibodies in the immune response to vaccination by anti-idiotypic antibodies mimicking the HMW MAA. The anti-tyrosinase antibody kinetic curves presented an initial increase in titres in five cases followed by decreasing titres; in two cases a constant decrease was noted. The inhibition assay demonstrated an increasing percentage of inhibition (range 17-100%) within 100-400 days of treatment. The titre of the anti-tyrosinase antibodies increased following the vaccination, then decreased--probably due to absorption of the antibodies to melanoma cells and normal melanocytes. A positive slope in the percentage of inhibition was roughly associated with a negative slope of anti-tyrosinase antibodies. In one case, a long-standing complete clinical response was accompanied by development of melanoma-associated hypopigmentation. Anti-B16 epitope antibodies had no role in the response to vaccination. The development of anti-tyrosinase antibodies in response to vaccination by anti-idiotypic antibodies mimicking another antigen may be explained by induction of non-specific polyclonal B lymphocytes activation, a well-recognized phenomenon in autoimmune disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca antagonist verapamil and tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) induce chromosomal aberrations in human lymphocytes.

Lymphocytes from 8 healthy donors were cultured for 3 days in the presence of phytohemagglutinin. Addition of the Ca antagonist verapamil or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased chromosomal aberrations in comparison with control cultures. The effects of TPA and verapamil were additive. Although the Ca ionophore A23187 had no effect per se, it did abolish the effect of verapamil. Five patients with supraventricular tachycardia were treated clinically with verapamil for 1 week. Their lymphocytes were cultured for 3 days in vitro in the presence of phytohemagglutinin. These lymphocytes showed higher chromosomal aberrations in comparison with lymphocytes isolated from the patients before treatment. The clinical significance is still unclear. We suggest that calcium ions may be necessary for the intactness of chromosomes of human lymphocytes and presumably of other cells.

Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell-mediated immunity.

Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell-mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed-type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 micrograms) or present in vitro (0.1 microgram/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell-mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.

Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with low doses of heparins.

The ability of activated T lymphocytes to penetrate the extracellular matrix and migrate to target tissues was found to be related to expression of a heparanase enzyme (Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Nature (Lond.). 310:241-243; Savion, N., Z. Fuks, and I. Vlodavsky. 1984. J. Cell. Physiol. 118:169-176; Fridman, R., O. Lider, Y. Naparstek, Z. Fuks, I. Vlodavsky, and I. R. Cohen. 1987. J. Cell. Physiol. 130:85-92; Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We found previously that heparin molecules inhibited expression of T lymphocyte heparanase activity in vitro and in vivo, and administration of a low dose of heparin in mice inhibited lymphocyte traffic and delayed-type hypersensitivity reactions (Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We now report that treatment with commercial or chemically modified heparins at relatively low doses once daily (5 micrograms for mice and 20 micrograms for rats) led to inhibition of allograft rejection and the experimental autoimmune diseases adjuvant arthritis and experimental autoimmune encephalomyelitis. Higher doses of the heparins were less effective. The ability of chemically modified heparins to inhibit these immune reactions was associated with their ability to inhibit expression of T lymphocyte heparanase. There was no relationship to anticoagulant activity. Thus heparins devoid of anticoagulant activity can be effective in regulating immune reactions when used at appropriate doses.

Vaccination against experimental autoimmune encephalomyelitis using a subencephalitogenic dose of autoimmune effector cells (1). Characteristics of vaccination.

We previously reported that rats could be vaccinated against EAE by inoculation with 10(7) anti-basic protein (anti-BP)-activated T cells raised as long-term lines. The activated T lines were irradiated (1,500 rads) to prevent them from causing EAE. We now report that a single inoculation of 10(4) or fewer cells of an activated anti-BP T-cell line did not cause clinical EAE but rather induced marked resistance to EAE produced by adoptive transfer of the anti-BP T cells. Resistance was less effective against EAE induced by active immunization to BP. Vaccination was immunologically specific, long lasting, and could be effected by various routes of administration.

Michaelis-Gutmann bodies in a healing brain infarct.

A case of Michaelis-Gutmann bodies in a healing brain infarct is described. Morphologically this was consistent with cerebral malacoplakia. There are only 6 previously reported cases of cerebral malacoplakia, and only 1 of them was a consequence of postpartum stroke. The histologic and ultrastructural features of the malacoplakia lesion are reported and compared with the previously reported cases.