Concomitant immune-related events in Wilson disease: implications for monitoring chelator therapy.

BACKGROUND AND AIMS: Current guidelines favor the use of chelating agents (d-penicillamine, trientine) in first line therapy of symptomatic Wilson disease patients. Development of chelator induced immunological adverse events are a concern especially under d-penicillamine therapy. This study assessed the prevalence of co-existing or therapy-related immune-mediated diseases in Wilson disease patients, and evaluated the role of antinuclear antibodies in therapy monitoring. METHODS: We retrospectively analyzed 235 Wilson disease patients. Medical regimens were classified and analyzed in relation to adverse events and antinuclear antibody courses. RESULTS: Coexisting immune-mediated diseases were evident in 19/235 (8.1%) patients, of which 13/235 (5.5%) had pre-existing autoimmune diseases. Six patients (2.6%) developed an autoimmune disease under therapy, all of them under long-term d-penicillamine treatment. Data relating to antinuclear antibody courses during treatment and adverse events were available for patients treated with d-penicillamine (n = 91), trientine (n = 58), and zinc salts (n = 58). No significant increase in antinuclear antibody titers in patients treated with d-penicillamine (16/91; 17.6%), trientine (12/58; 20.7%), and zinc (7/58; 12.1%) were found. CONCLUSION: Under long-term d-penicillamine therapy a minority of patients developed immune-mediated disease. Elevations in antinuclear antibodies were found frequently, but no correlations were evident between increases in antinuclear antibodies and the development of immune-mediated diseases or medical regimes. Thus, the value of antinuclear antibodies for monitoring adverse events under chelator therapy seems to be limited.

[D-penicillamine-induced pemphigus: changes in anti-32-2B immunostaining patterns]. / Autonomisation d'un pemphigus induit par la D-pénicillamine: évolution de l'immunomarquage par l'anticorps anti-32-2B.

BACKGROUND: It has been reported that D-penicillamine causes pemphigus that is typically superficial. Immunostaining with monoclonal anti-32-2B antibody targeting desmoglein 1 and 3 can help differentiate between drug-induced and classical auto-immune pemphigus. Absence of specific staining militates in favour of drug-induced pemphigus whilst positive staining suggests an auto-immune aetiology that is ongoing despite discontinuation of drug therapy. PATIENTS AND METHODS: A 59-year-old male patient was referred for management of superficial pemphigus 1 year after starting D-penicillamine treatment for scleroderma. The diagnosis of pemphigus was confirmed histologically (intra-epidermal cleavage, acantholysis and perikeratinocytes, deposition of IgG and complement C3). Immunochemical staining with anti-32-2B antibody was initially normal, in keeping with drug-induced pemphigus. Despite discontinuation of D-penicillamine, pemphigus recurred in 2008. A further skin biopsy was undertaken and anti-32-2B staining was abnormal, which is consistent with auto-immune pemphigus. DISCUSSION: Numerous cases of drug-induced pemphigus have been described in the literature. In approximately half of all cases, the pemphigus recedes after cessation of the causative drug. However, there have been no previous reports that changes over time in the immunostaining with anti-32-2B antibodies can mirror a change in form of pemphigus from a drug-induced type to an idiopathic type as well as the associated clinical feature of persistence after drug withdrawal. CONCLUSION: Normal staining with anti-32-2B antibody is associated with a favourable prognosis as regards resolution of drug-induced pemphigus. When, as in this case, status changes to abnormal staining, there is a risk that the pemphigus may become chronic despite discontinuation of therapy.

T cell responses to D-penicillamine in drug-induced myasthenia gravis: recognition of modified DR1:peptide complexes.

The anti-rheumatoid drug D-penicillamine (D-pen) has a reactive sulfhydryl group capable of modifying self antigens, and can provoke typical autoantibody-mediated myasthenia gravis (MG), especially in DR1+ individuals. We have selected T cell clones from one such patient that were highly specific for D-pen but not its L-isomer or D-cysteine. Moreover, they were restricted to HLA-DR1, had a Th1 phenotype and used TCR V alpha4.1, V beta6.1. They responded well to blood mononuclear cells prepulsed with D-pen either in the absence of serum or after chloroquine treatment, but not to autologous D-pen-pulsed B cell lines. Thus, D-pen may directly couple to distinctive peptides resident in surface DR1 molecules on circulating macrophages or dendritic cells.

Drug-protein conjugates--XV. A study of the disposition of D-penicillamine in the rat and its relationship to immunogenicity.

The disposition of [14C]D-penicillamine (PA) was investigated in vitro and in vivo with male Wistar rats. Irreversible binding of [14C]PA to isolated rat plasma proteins in vitro reached a maximum of 20.6% of total radioactivity at 6 hr. Irreversibly bound [14C]PA could be dissociated with dithiothreitol, demonstrating that conjugation was via disulphide linkage. Three hours after i.v. administration of [14C]PA (27 mumol/kg) to rats 100% of plasma radioactivity was irreversibly bound, representing approximately 3.5% of the dose. Further studies on the disposition of PA-plasma protein conjugates showed that dissociation occurred readily in vivo: the plasma half-life of the conjugate was approximately 3 hr. Free [14C]PA was the major urinary metabolite after administration of both free and conjugated drug. These studies show that the disposition of PA is similar to that reported for the structurally related sulphydryl drug captopril (CP). Free PA (340 mumol/kg and 3.4 mmol/kg) administered i.p. and i.m. daily for 4 days at one monthly intervals, and also PA-KLH conjugate (100 micrograms/rat) administered by single i.p. injection at monthly intervals with and without Freund's complete adjuvant, failed to induce PA-specific IgG or IgM antibody responses detectable by ELISA. In contrast, CP (270 mumol/kg), administered by the same protocol as free PA, induced a CP-specific IgG antibody response after the third series of monthly injections. These data suggest that the difference in immunogenicity between PA and CP arises from a difference in the intrinsic immunogenicity of the haptens, rather than from their disposition.

Striational autoantibodies: quantitative detection by enzyme immunoassay in myasthenia gravis, thymoma, and recipients of D-penicillamine or allogeneic bone marrow.

Striational autoantibodies (StrAb) are a useful serologic marker of thymoma in patients with myasthenia gravis (MG). We compared a standard immunofluorescence method with a new enzyme immunoassay (EIA) for detection of StrAb. Retrospective testing of 264 stored sera by the two methods yielded well-correlated results (58 sera were positive by both assays; r = 0.8). For 104 patients with spontaneously acquired MG or thymoma, results were 100% concordant, of which 53% were positive. For 34 recipients of D-penicillamine, StrAb were found in 15% by EIA and in 6% by immunofluorescence. StrAb were detected in two of four bone marrow recipients by EIA and in one by immunofluorescence. Prospective testing of 434 fresh sera (of which 49 were positive by the two methods) yielded discordant results in only 4. Serial EIA quantitation of StrAb in two patients with MG and thymoma proved useful in monitoring immunosuppressant therapy and in a third patient predicted recurrence of the tumor. A high prevalence of StrAb was detected by both assays in elderly patients with spontaneous MG, but StrAb were more readily quantifiable by EIA. The EIA method proved to be highly sensitive and specific for detecting StrAb in patients with thymoma with and without MG, in patients treated with D-penicillamine, and in those with graft-versus-host disease after bone marrow transplantation.

D-penicillamine-induced autoantibodies in a mouse model.

OBJECTIVE: We have previously shown that the administration of D-penicillamine (D-PEN) to patients with rheumatoid arthritis induces circulating insulin autoantibodies (INSAAB). In order to gain further insight into such immune responses, we measured a battery of circulating autoantibodies in 4 strains of mice receiving D-PEN: C57BL/KsJ, BALB/c, C3H/HeJ, and C57BL/6. These rodents groups differ in their degree of susceptibility to streptozotocin (STZ)-induced immune diabetes (SIMD), which is high in the first 2 strains, and mild and nil in the third and fourth, respectively. METHODS: Randomly assigned animals from each group were given a weekly subcutaneous (SC) injection of either D-PEN 1 mg, D-PEN 3 mg, or solvent (PBS) for a period of 4 weeks. Serum levels of antibodies to insulin, single stranded DNA (ssDNA), thyroglobulin, and cardiolipin were measured weekly. RESULTS: Only the C57BL/KsJ and C3H/HeJ mice reacted to D-PEN administration. When compared to the pre-treated and solvent-treated mice, D-PEN 1 mg, and to a lesser degree D-PEN 3 mg, induced elevation of antibodies to insulin and to ssDNA in C57/KsJ mice (p < 0.001), while only ssDNA antibodies were detected in the C3H/HeJ mice (p < 0.0001 for D-PEN 1 mg; p < 0.05 for D-PEN 3 mg). D-PEN had no effect on the level of antibodies to cardiolipin or to thyroglobulin in any of the mice. CONCLUSIONS: This study showed that D-PEN induces an antigen(s)-specific humoral response only in mice already inherently prone to autoimmunity. This model suggests that the activation of autoimmunity by environmental factors is probably facilitated by genetic background, and might partly explain the diversity of autoimmune manifestations in D-PEN-treated patients.

Effect of selected immunoregulatory agents on low-grade contact sensitivity.

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57Bl/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062-1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31-5.0 mg/kg intravenously), penicillamine (6.25-25 mg/kg, subcutaneously), chloroquine (6.25-25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125-1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.

Progressive systemic sclerosis complicated with immune thrombocytopenia during D-penicillamine therapy.

Immune thrombocytopenia is a rare complication of progressive systemic sclerosis (PSS). A 47-year-old female with PSS treated with D-penicillamine developed immune thrombocytopenia, which promptly responded to prednisolone and withdrawal of D-penicillamine. Platelet-associated IgG was elevated and the bone marrow megakaryocyte count was normal. There was an inverse relationship between the level of platelet-associated IgG and the platelet count. A lymphocyte stimulation test sensitized by D-penicillamine was positive. The present case suggests that immune thrombocytopenia may be regarded as one of the D-penicillamine-related immune abnormalities. To our knowledge, its association with PSS has never been reported.

[Detection of antibodies against D-penicillamine. 2. Experimental animal proof of production of antibodies against D-penicillamine using indirect immunofluorescence with a DASS system]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 2. Mitteilung: Nachweis tierexperimentell erzeugter Antikörper gegen D-Penicillamin durch indirekte Immunfluoreszenz mittels DASS-System.

Antibodies against D-penicillamine conjugates produced in animals be proved by indirect immunofluorescence by means of the DASS system (defined antigen substrate spheres). The artificial substrate (Sephadex G-25) has not been bound to the D-penicillamine conjugates used for immunization, but only to D-penicillamine, which justifies the assumption that antibodies against D-penicillamine could be registered and proved by the immunofluorescence test mentioned.

[The detection of antibodies against D-penicillamine. 1. Coupling of D-penicillamine to various carriers and generation of antibodies against D-penicillamine conjugate]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 1. Mitteilung: Kopplung von D-Penicillamin an verschiedene Träger und Erzeugung von Antikörpern gegen D-Penicillamin-Konjugate.

D-Penicillamin as 2-substituted 5,5-Dimethyl-thiazolidine-4-carbonic acid has been bound to poly-L-lysin, human serum albumin and bovine gamma globulin by means of a water-soluble carbodiimid. The anti-bodies generated at animal tests by immunization of guinea pigs had been proved by means of the radial immune diffusion acc. to Ouchterlony [10].

[The detection of antibodies against D-penicillamine. 3. Detection of antibodies against D-penicillamine in the serum of patients with Wilson's disease treated with D-penicillamine--preliminary results]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 3. Mitteilung: Nachweis von Antikörpern gegen D-Penicillamin im Serum von Patienten mit Morbus Wilson unter der Behandlung mit D-Penicillamin - Vorläufige Ergebnisse.

121 serum samples from 54 patients with Wilson's disease were tested for antibodies against D-penicillamine by indirect immunofluorescence (DASS-system). IgG antibodies were found in 44 serum samples from 16 patients (31% of all patients). The incidence of serum antibodies was higher in patients with side effects during therapy with D-penicillamine (10 of 13 patients 77%) compared to 6 of 39 15% in patients without side effects. The titre of antibodies was higher in patients with side effects. The antibodies bound complement demonstrated by double immunofluorescence. These observations indicate that complement binding IgG antibodies to D-penicillamine are involved in pathogenesis of side effects during therapy with D-penicillamine.

Proteinuria in D-penicillamine-treated rheumatoid arthritis.

The incidence of proteinuria in D-penicillamine-treated rheumatoid arthritis has been between 10% and 20% in the 1st year of therapy; afterward, few develop proteinuria. Incidence is related to both quantity of penicillamine administered and inherent response variability. About 1/3 of those with proteinuria may proceed to a nephrotic syndrome if therapy is continued. Light microscopy of renal biopsy tissue shows only minimal glomerular changes. Electron microscopy reveals sub-epithelial electron-dense deposits and fusion of epithelial cell processes. Immunofluorescence microscopy demonstrates granular capillary wall deposits of IgG and C3.

Immunobiology of D-penicillamine.

D-penicillamine holds the key to a better understanding of autoimmunization and the pathogenesis of autoimmune disease. Analysis of its mode of action is complicated by its multiplicity of effects. In respect to anti-acetylcholine receptors and myasthenia gravis, the major effect may be at the level of immunoregulation and/or immunogenicity. Anti-striated muscle antibody is much more common and is influenced by the HLA antigen of the patient. Thus, HLA-linked immune response genes may be involved.

Differential requirement for CD28/CTLA-4-CD80/CD86 interactions in drug-induced type 1 and type 2 immune responses to trinitrophenyl-ovalbumin.

The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-gamma, TNF-alpha, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.

A western blot approach to detection of human plasma protein conjugates derived from D-penicillamine.

OBJECTIVES: To develop and apply an immunochemical approach to the study of drug-plasma protein conjugates derived from the anti-arthritic drug D-penicillamine (DP). METHODS: An antiserum with specificity for protein-conjugated DP was raised in a rabbit. Plasma samples from patients receiving DP or from incubations of isolated normal plasma with DP were analysed for DP-derived conjugates by Western blotting using the anti-drug antibody. RESULTS: A single DP-positive protein band was detected in plasma samples from 15/16 patients with rheumatoid arthritis receiving DP but in none of 20 patients of similar disease status who had not taken DP. The positive band appeared in patients' plasma during the course of treatment with DP. It was seen under nonreducing but not reducing conditions indicating that the drug is disulphide linked to the protein. The drug-modified protein migrated to a position intermediate between the trailing edge of albumin and the leading edge of transferrin (both non-reduced) suggesting a molecular weight of between 66 and 77 kDa. Incubations of normal human plasma, but not purified albumin or transferrin, with low concentrations of DP generated the same distinct band plus several less intense DP-positive bands. CONCLUSIONS: Drug-plasma protein conjugates derived from DP in vivo and in vitro can be detected immunochemically by the Western blot method. Theories of DP immunotoxicity have implicated antigenicity of the drug, but this is the first immunochemical demonstration of a potential DP-derived antigen in human plasma. The method we describe may have application to studies of the relationship between DP antigenicity and toxicity.

A T-cell response to the anti-arthritic drug penicillamine in the mouse: requirements for generation of the drug-derived antigen.

Mice primed with the anit-arthritic drug D-penicillamine (DP) developed DP-specific T cells in the draining lymph nodes (DLN) which responded to drug-haptenated stimulator cells, but not to untreated control cells nor to free drug, in in vitro proliferation assays. The responder cells were CD4+ and the response was major histocompatibility complex (MHC) class II restricted. The conditions required to generate efficient stimulator cells for in vitro proliferation assays were investigated. Drug-haptenated syngeneic spleen cells, but not thymocytes, were able to stimulate T cells from DP-sensitized mice. However, prolonged incubations of spleen cells with DP were required to generate the drug-derived T-cell antigen. Further experiments revealed that the generation of a DP-derived antigenic determinant for T cells did not require intracellular processing, as stimulator cells pretreated with fixative or lysosomotropic agents before drug haptenation were as effective as untreated DP-haptenated cells in stimulating the responder cells to proliferative in vitro. These findings show that the protein-reactive drug DP can generate a cellular antigen that is capable of stimulating a T-cell response. Furthermore, the generation of this antigen appears to bypass conventional antigen processing, suggesting perhaps a direct chemical modification of cell surface molecules that are involved in immune recognition. This process may underlie adverse reactions to DP that are believed to be mediated by the cellular immune system.