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.

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.

Contribution of Va24Vb11 natural killer T cells in Wilsonian hepatitis.

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.

[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.

Treatment of Wilson's disease with zinc: XIV. Studies of the effect of zinc on lymphocyte function.

Although administration of zinc to human subjects has been reported to interfere with lymphocyte function, this single report has never been confirmed or refuted. We have developed zinc as a lifelong therapy for patients with Wilson's disease. Interference with lymphocyte function occurring as a side effect of zinc therapy could produce serious problems in our patients. We evaluated lymphocyte mitogenic response and natural killer cell activity in patients with Wilson's disease treated for 5 years or longer with zinc, in comparison with normal controls, and found no differences. In a second study, we evaluated these same parameters in patients with Wilson's disease before and after 1 year of zinc therapy, and again found no significant differences. We have seen no indications of immune suppression or increased susceptibility to infections in our patients, who have now been treated with zinc for up to 15 years. We conclude that any side effects from compromised lymphocyte function caused by administration of zinc are not of concern to patients with Wilson's disease.

Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line.

A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of 125I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

Patterns of hepatocyte injury in man.

Three patterns of hepatocyte injury in man, direct, immunological, and cholestatic, are described. The characteristics of the direct pattern are predominantly mitochondrial damage, central (zone 3) necrosis, and, usually, fatty change. It can be subdivided into the alcohol type (also seen with obesity, in diabetes, as a reaction to perhexiline, in Wilson's disease, and in Indian childhood cirrhosis) and the Reye's syndrome type (also seen with tetracycline toxicity, fatty liver of pregnancy, and cytotoxic drugs). Reactive drug metabolites, metal poisoning, and anoxia are also associated with the direct pattern of hepatocyte injury. The immunological pattern is characterised by damage to cell membranes with piecemeal necrosis of periportal (zone 1) hepatocytes and mononuclear-cell infiltration. Examples include chronic active hepatitis, primary biliary cirrhosis, and drug reactions such as those to halothane. In the cholestatic pattern there is disturbance of the bile-secretory mechanism with retention of bile within the hepatocytes. Cholestatic liver injury may be intrahepatic, as in sex-hormone cholestasis, or extrahepatic, as in choledocholithiasis or carcinoma of the bile ducts. Identification of the type of hepatocyte injury is valuable in diagnosis, in assessing prognosis, and in selecting treatment.

[immunohistologic findings of kidney biopsies in Wilson's disease]. / Immunhistologische Befunde an Nierenbiopsien bei Morbus Wilson.

12 patients with Wilson's disease were examined by means of renal biopsy. In 9 of these patients a slight (4 patients) or a strong proteinuria (5 patients) appeared during the therapy with D-penicillamine. A biopsy was performed in 3 patients before the beginning of the therapy with D-penicillamine. The patients with severe proteinuria showed the largest immunohistological changes (partly distinct immune complex nephritis with epimembranous IgG- and complement-(C3-) depositions -- electronoptically membranous glomerulonephritis. In rebiopsy after a stoppage of the medicament of 1 year only a slight tendency to improvement was to be recognized.

[Wilson's liver disease in children and adolescents (author's transl)]. / Die hepatische Manifestationsform des Morbus Wilson im Kindes- und Jugendalter.

Hepatic symptoms are usually the first in Wilson's disease of children and adolescents, while neurologic symptoms and the corneal ring are still missing. Liver lesions due to copper accumulation may develop throughout years without clinical symptoms or biochemical abnormalities. Hemolytic jaundice or gastrointestinal bleeding are the presenting symptoms in some cases. In spite of being a rare syndrom Wilson's disease ought to be considered after hepatitis B or autoimmune liver disease have been excluded as causes of juvenile cirrhosis of the liver. If life-long treatment with D-penicillamin is started in an early stage of Wilson's disease, prognosis is rather good.

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.

On the defect of synthesis ceruloplasmin in the liver polyribosomes in Wilson's disease.

Comparative immunochemical analysis of ceruloplasmin-synthesizing polyribosomes in liver biopsies from control subjects and homozygous carriers of the Wilson's mutation was performed. According to I125-antibody binding data, the amount of ceruloplasmin-forming liver polysomes in patients with Wilson's disease was 10--20 times lower than that in non-Wilson patients. Correspondingly, the pulse labeling of ceruloplasmin polypeptides was decreased several-fold in the cell-free liver preparations from patients with Wilson's disease.