Wilson's Disease-Crossroads of Genetics, Inflammation and Immunity/Autoimmunity: Clinical and Molecular Issues.

Wilson's disease (WD) is a rare, autosomal recessive disorder of copper metabolism caused by pathogenic mutations in the ATP7B gene. Cellular copper overload is associated with impaired iron metabolism. Oxidative stress, cuproptosis, and ferroptosis are involved in cell death in WD. The clinical picture of WD is variable. Hepatic/neuropsychiatric/other symptoms may manifest in childhood/adulthood and even old age. It has been shown that phenotypic variability may be determined by the type of ATP7B genetic variants as well as the influence of various genetic/epigenetic, environmental, and lifestyle modifiers. In 1976, immunological abnormalities were first described in patients with WD. These included an increase in IgG and IgM levels and a decrease in the percentage of T lymphocytes, as well as a weakening of their bactericidal effect. Over the following years, it was shown that there is a bidirectional relationship between copper and inflammation. Changes in serum cytokine concentrations and the relationship between cytokine gene variants and the clinical course of the disease have been described in WD patients, as well as in animal models of this disease. Data have also been published on the occurrence of antinuclear antibodies (ANAs), antineutrophil cytoplasmic antibodies (ANCAs), anti-muscle-specific tyrosine kinase antibodies, and anti-acetylcholine receptor antibodies, as well as various autoimmune diseases, including systemic lupus erythematosus (SLE), myasthenic syndrome, ulcerative colitis, multiple sclerosis (MS), polyarthritis, and psoriasis after treatment with d-penicillamine (DPA). The occurrence of autoantibodies was also described, the presence of which was not related to the type of treatment or the form of the disease (hepatic vs. neuropsychiatric). The mechanisms responsible for the occurrence of autoantibodies in patients with WD are not known. It has also not been clarified whether they have clinical significance. In some patients, WD was differentiated or coexisted with an autoimmune disease, including autoimmune hepatitis or multiple sclerosis. Various molecular mechanisms may be responsible for immunological abnormalities and/or the inflammatory processes in WD. Their better understanding may be important for explaining the reasons for the diversity of symptoms and the varied course and response to therapy, as well as for the development of new treatment regimens for WD.

Inflammatory cytokines expression in Wilson's disease.

BACKGROUND: Wilson's disease (WD) is an autosomal recessive inherited disorder of copper (Cu) metabolism. Inflammation is a self-defensive reaction aimed at eliminating or neutralizing injurious stimuli, and restoring tissue integrity. Copper deposition may lead to inflammation in the organs and tissues of WD patients. OBJECTIVE: The aim of this study was to compare the plasma levels of inflammatory cytokines in patients with WD and healthy group, and also to assess whether inflammatory cytokines affects the clinical manifestation of WD. METHODS: Ninety-nine patients with WD and 32 controls were recruited for this study. Ray Biotech antibody microarray was used to detect the levels of plasma inflammatory cytokines. RESULTS AND CONCLUSION: Our results showed significant increase in T helper (Th) 1 cells (IL-2, TNF-α, and TNF-ß), Th2 cells (IL-5, IL-10, and IL-13), and Th17 (IL-23) (p < 0.05). Higher plasma Th 1 cells (IL-2, TNF-α, and TNF-ß), Th 2 cells (IL-13), and Th 17 (TGF-ß1, IL-23) levels were found in neurological patients compared with control groups (p < 0.01). Besides, we found Th 1 cells (TNF-α and TNF-ß), Th 3 (TGF-ß1), and Th 17 (IL-23) levels were significantly higher in hepatic and neurological patients (p < 0.05). In addition, the higher Th1 cells (IL-2, TNF-α, and TNF-ß), Th2 cells (IL-13), and Th17 (TGF-ß1, IL-23) and the course of WD were associated with the severity of the neurological symptoms for WD patients. Altogether, our results indicated that dysregulation of cytokines, mainly increased expression of cytokines and chemokines, occurred in WD patients.

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.

MiADMSA abrogates chronic copper-induced hepatic and immunological changes in Sprague Dawley rats.

Wilson disease (WD) is an autosomal-recessive disorder associated with the impaired copper metabolism, resulting in hepatic and neurologic manifestations. D-Pencillamine (DPA) is a first-line of treatment however, monoisoamyl 2, 3-dimercaptosuccinic acid (MiADMSA), is gaining recognition recently as a future chelating agent of choice. We evaluated the effects of MiADMSA against copper-induced (20 mg/kg, orally, once, daily for 16 weeks) hepatic and immunological changes in the male Sprague Dawley (SD) rats. Copper overload increased the levels of pro-oxidant and concurrently decreased the levels of antioxidant enzymes in the liver. Increased oxidative stress triggered the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) in the liver and down-regulated the anti-inflammatory cytokine IL-4. Altered liver function parameters as well as serum immunoglobulins' (IgG, IgA, IgE, and IgM) levels, were also noted. MiADMSA treatment restored most of copper altered biochemical and immunological changes. Further, the histopathological changes proved that MiADMSA treatment ameliorated copper induced hepatic injury. Infra red spectra of liver tissue indicated shift in the characteristic -OH peak during copper exposure while the shifting came to normal in MiADMSA administered rat liver. We conclude that MiADMSA could be a promising antidote for the chronic copper toxicity and possibly in the clinical management of WD.

Reduced Neutrophil Extracellular Trap (NET) Formation During Systemic Inflammation in Mice With Menkes Disease and Wilson Disease: Copper Requirement for NET Release.

Neutrophil extracellular traps (NETs) contribute to pathological disorders, and their release was directly linked to numerous diseases. With intravital microscopy (IVM), we showed previously that NETs also contribute to the pathology of systemic inflammation and are strongly deposited in liver sinusoids. Over a decade since NET discovery, still not much is known about the metabolic or microenvironmental aspects of their formation. Copper is a vital trace element essential for many biological processes, albeit its excess is potentially cytotoxic; thus, copper levels are tightly controlled by factors such as copper transporting ATPases, ATP7A, and ATP7B. By employing IVM, we studied the impact of copper on NET formation during endotoxemia in liver vasculature on two mice models of copper excess or deficiency, Wilson (ATP7B mutants) and Menkes (ATP7A mutants) diseases, respectively. Here, we show that respective ATP7 mutations lead to diminished NET release during systemic inflammation despite unaltered intrinsic capacity of neutrophils to cast NETs as tested ex vivo. In Menkes disease mice, the in vivo effect is mostly due to diminished neutrophil infiltration of the liver as unmutated mice with a subchronic copper deficiency release even more NETs than their controls during endotoxemia, whereas in Wilson disease mice, excess copper directly diminishes the capacity to release NETs, and this was further confirmed by ex vivo studies on isolated neutrophils co-cultured with exogenous copper and a copper-chelating agent. Taken together, the study extends our understanding on how microenvironmental factors affect NET release by showing that copper is not a prerequisite for NET release but its excess affects the trap casting by neutrophils.

Pros and cons of immunochemical and enzymatic method in the diagnosis of Wilson's disease.

BACKGROUND: Immunochemical method of measuring Ceruloplasmin (Cp) levels for the diagnosis of Wilson's disease has replaced enzymatic method for the main reason of being more sensitive and quantitative. SETTINGS AND DESIGN: In this study, we compared both the methods for various factors such as sensitivity, specificity and the time consumed in the diagnosis of Wilson's disease. MATERIALS AND METHODS: Serum samples from patients (n=33) with a provisional diagnosis of Wilson's disease were analyzed for Cp levels by enzymatic copper oxidase method and immunochemical method using polyclonal antibodies specific to Cp embedded in agar. STATISTICAL ANALYSIS: Pearson's regression analysis was performed to compare the two methods. RESULTS: The mean Cp obtained by immunochemical method is 5.87 mm +/- 1.17 and by enzymatic method, it is 0.37 (Optical Density) +/- 0.136. Pearson's Regression analysis of the measurements showed a good correlation with an 'r' value of 0.765 between the two methods. CONCLUSION: A good correlation indicated that these two tests are comparable and thus both these methods can be used together for a definitive and better diagnosis of Wilson's disease.

Immunological observations on patients with Wilson's disease.

In 19 patients with Wilson's disease we found an increased humoral immune response, i.e. a higher level of IgG and IgM, a higher titre of antibodies against Kunin's CA antigen and a depressed cell-mediated immunity i.e. a lower response to DNCB and E. coli in skin tests, lower lymphocyte transformation when stimulated by Con A, PPD, Candida albicans and streptokinase and a lower production of macrophage migration inhibition factor. The changes observed in the group of patients with liver cirrhosis caused by other facotrs than Wilson's disease were similar but less pronounced. We also found that leukocytes of patients with Wilson's disease have an impaired bactericidal activity and that copper ions have an inhibitory effect on some tests for cell-mediated immunity. It seems probable that immunological abnormalities in Wilson's disease are caused by liver cirrhosis but we cannot exclude an inhibitory effect of copper ions upon the immune response and an associated effect upon leukocyte metabolism.

[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.

[Cytochemical studies of peripheral white blood cells in Wilson's disease]. / Badania cytochemiczne w krwinkach bialych krwi obwodowej w chorobie Wilsona

Using cytochemical methods the authors studied the activity of certain lysosomal enzymes and cytochrome oxidase in peripheral blood leucoytes in 22 patients with Wilson's disease. The control group comprised 50 healthy blood donors. It was found that the activity of acid phosphatase in the lymphocytes of patients was higher than in controls, the mean indices being respectively 90.50 +/- 8.95 and 60.38 +/- 3.95. The activity of beta-glucuronidase was found to be lower in the lymphocytes of patients, the mean value was 25.10 +/- 8.59 in patients and 64.91 +/- 5.78 in controls. The activity of cytochrome oxidase was lower in the granulocytes of patients with Wilson's disease than in controls, the mean values being 54.5 +/- 12.14 and 156 +/- 15.41 respectively. The activity of acid phosphatase in granulocytes as well as that of non-specific esterase in lymphocytes was similar in both groups. Decreased antigen degradation in Wilson's disease may be due not only to liver cirrhosis but also to disturbances in the metabolism of white blood cells, including, among others, decreased activity of cytochrome oxidase. The rise of the activity of acid phosphatase and reduced activity of beta-glucuronidase indicate chronic antigenic stimulation of lymphoid system.

[Relation between serum immunoglobulin levels and anticerebral antibodies, detected by the Coon method, in hepatolenticular degeneration]. / O zwiazku poziomu immunoglobulin surowiczych i liczby przeciwcial przeciwmózgowych, wykrywanych metoda Coonsa u chorych na zwyrodnienie watrobowo-soczewkowe.

Antibodies to brain tissue were determined by indirect immunofluorescence of Coons in 23 patients with hepatolenticular degeneration. In 14 patients the levels of immunoglobulins A, G and M were determined as well. Control investigations were done in 37 donors. Positive results of Coons reaction with bright fluorescence of nervous tissue antigens were obtained in patients with increased serum immunoglobulin levels. Qualitative differences were obtained also in the reaction of Coons with sera from various patients. These data evidence that immunoglobulin F (ab)2 fragments as well as Fc fragments participate in the immunofluorescent reaction which calls for caution in the interpretation of the results of this method.

[T-rosette test in Wilson's disease]. / Zachowanie sie testu T rozety i odczynów skórnych w chorobie Wilsona

Twenty-three patients with Wilson's disease were studied. Using the rosette formation test it was found that the count of T-lymphocytes in the peripheral blood of these patients (29,77% +/- 12,58) was lower than in the control group (61,68% +/- 13,14). Using skin tests for demonstration of delayed hypersensitivity it was noted that in the group of patients the frequency of positive reaction with Candida albicans antigen was lower and these patients showed also less frequently positive reactions after DNCB immunization. The obtained results indicate that in Wilson's disease the functions of T-lymphocytes are distrubed. This may be the cause of previously observed hyperactivity of B-lymphocytes.

[Vascular purpura associated with chronic diffusive hepatic pathologies]. / Sosudistaia purpura, assotsiirovannaia s khronicheskimi diffuznymi zabolevaniiami pecheni.

The study results related with the rate of vascular purpura in 660 patients with different-type chronic diffusive hepatic pathologies both of the viral and other natures are described in the paper. The main regularities characterizing the phenomenon (spread and possible cause of purpura development) are defined. According to an analysis of actual materials, dermal vasculitis is not an exclusive feature of virus-associated hepatitis or liver cirrhosis. Vascular purpura can be regarded, on the basis of the obtained data, as a universal sign typical of any liver pathologies.

[Molecular genetic and biochemical studies of human inherited diseases]. / Molekuliarno-geneticheskoe i biokhimicheskoe izuchenie nasledstvennykh zabolevanii cheloveka.

The paper gives the results of complex studies of human inherited diseases, which involved reverse genetics for the analysis of mutant genes, biochemical and immunological studies of anomalous proteins, the products of these genes. It describes the main results of the studies which used this complex approach to examining the etiology and pathogenesis of Wilson's disease, alpha 1-antitrypsin deficiency, familial hypercholesterolemia and cystic fibrosis.

[Clinico-immunologic correlations and various characteristics of the lymphocyte receptors in hepatocerebral dystrophy]. / Kliniko-immunologicheskie sopostavleniia i nekotorye osobennosti retseptorov limfotsitov pri gepatotserebral'noi distrofii.

Clinical and immunological studies were performed in 45 patients with HCD. A group of patients was investigated by conventional immunology tests. spontaneous and active rosette formation, theophylline test, immunofluorescent B-lymphocyte investigation, IgA, IgM, IgG and circulating immune complexes (IC) levels measurement. Monoclonal antibodies were used to count T- and B-lymphocytes, T-helpers and T-suppressors in another group. [3H]-spiroperidol binding to blood lymphocytes was studied in 28 patients. Immune indices were found to be related to the disease duration, degree of visceral deterioration and the duration of Cu-depleting drugs administration. After a prolonged treatment with D-Penicillamin or Cuprenil immune indices returned to normal levels while the count of theophylline-sensitive lymphocytes remained unchanged. The mechanisms of metabolic and immune interrelations in HCD are discussed.

[Features of the effect of the sera of patients with hepatocerebral dystrophy on nerve tissue in vivo and in vitro]. / Osobennosti vliianiia syvorotok bol'nykh gepatotserebral'noi distrofiei na nervnuiu tkan' in vivo i in vitro.

Electron microscopy of normal rabbit brain, conducted after the animals had been given intracerebral injection of serum obtained from patients with hepatocerebral dystrophy revealed some peculiarities of the damaging effect of such serum on the brain. Incubation of neural tissue culture with such serum resulted in appearance of hypertrophic astrocytes, type II Alzheimer's cells and Opalski's cells combined with the intact synthesis of an astrocyte specific antigen--an acid protein of astrocytic fibrility--detected by Coons' immunofluorescent test.

[Possibility of using the indirect fluorescent antibody technic to analyze immunoglobulins in certain neurogenetic diseases]. / O vozmozhnosti ispol'zovaniia metoda nepriamoi immunofliuorestsentsii dlia analiza immunoglobulinov pri nekotorykh neirogeneticheskikh zabolevaniiakh.

Results of examining the sera of 11 normal donors and 37 patients with various neutogenetic diseases using, the Coons indirect immunofluorescence technique are presented. The patients' immunoglobulins could be better fixed on nervous tissue components than those of the control donors. This seems likely to be due to both a rise in the content of serum immunoglobulins and accumulation of specific anticerebral antibodies in the patients' blood. It is suggested that in all cases of using indirect immunofluorescence the quantitative determination of immunoglobulins and serum titration should be carried out.

[Quantitative evaluation of the T- and B-systems of immunity in various hereditary diseases of the nervous system]. / Kolichestvennaia otsenka T- i B-sistem immuniteta pri nekotorykh nasledstvennykh zabolevaniiakh nervnoí sistemy.

In 37 patients with some hereditary diseases of the nervous system (deforming muscular dystonia, hepatocerebral dystrophy, essential tremor, etc.), as well as in 22 healthy subjects (donors), the percentage and the absolute content of the T- and B-lymphocytes in the blood were determined. Use was made of the reactions of spontaneous, active, and complementary rosette formation and determination of the B-cells from superficial immunoglobulins. In all the hereditary diseases listed a drop of the content of thymus-dependent lymphocytes and an increase of the capacity of lymphocytes for complementary rosette formation were revealed. The data obtained can be, probably, interpreted as evidences of a secondary immunodeficient state, possibly, caused by metabolic disturbances which are known to play an essential role in the pathogenesis of hereditary diseases of the nervous system.

A study of oxidative stress, cytokines and glutamate in Wilson disease and their asymptomatic siblings.

BACKGROUND: Free copper in Wilson disease (WD) is toxic and may reduce antioxidant, increase oxidative stress marker and thereby cytokine release and excitotoxic injury, but there is paucity of studies in humans. We report oxidative stress markers, cytokines and glutamate in neurologic WD and correlate these with their clinical severity, laboratory findings and extent of Magnetic resonance imaging (MRI) changes. METHODS: 29 patients with neurologic WD and 9 asymptomatic WD siblings were included and their clinical, treatment history, disease severity, biochemical findings and MRI changes were noted. Glutathione (GSH), total antioxidant capacity (TAC) and malonodialdehyde (MDA) were measured by spectrophotometer, cytokines by cytokine bead array and glutamate by the fluorometer. RESULTS: In WD patients, the glutathione (mean±SEM, 2.20±0.06 vs. 2.73±0.04mg/dl, P<0.001) and TAC (1.70±0.03 vs. 2.29±0.02 Trolox_Eq_mmol/l, P<0.001) were reduced, and MDA and glutamate (23.93±0.54 vs. 19.96±0.27µmol/l; P<0.001) were increased (4.7±0.11 vs. 3.03±0.52nmol/ml, P<0.001) compared to controls. The serum IL6 {median (IQRs), 9.42(10.92) vs. 5.2(5.34) pg/ml; P=0.001}, IL8 {12.37(10.92) vs. 5.63(5.52) pg/ml; P<0.001}, IL10 {8.33(8.3) vs. 2.05(1.37) pg/ml; P=0.001} and TNFα {6.14(8.95) vs. 3.61(3.58) pg/ml; P<0.001} were also increased in WD patients compared to controls. These changes were more marked in the neurologic WD compared to asymptomatic WD and in the untreated compared to treated patients. TAC correlated with duration of illness, serum free copper, 24hour urinary copper and serum ceruloplasmin, and glutamate with MDA, TNFα, ceruloplasmin and 24-hour urinary copper. CONCLUSIONS: In WD patients, antioxidants are reduced and MDA, cytokines and glutamate are increased which are more marked in symptomatic neurologic WD than asymptomatic patients.