Liver-specific Lipoprotein Research Articles

The clinical significance of serum ferritin in pediatric non-alcoholic Fatty liver disease.

PurposeNon-alcoholic fatty liver disease (NAFLD) in children has become an important public health issue because of its high prevalence and severity. Several noninvasive methods for estimating NAFLD are under investigation. We aimed to evaluate the usefulness of serum ferritin as a biomarker of severity of pediatric NAFLD patients.MethodsA total of 64 NAFLD patient were enrolled from Severance Children's Hospital from March 2010 to February 2013. Serum ferritin levels, liver related laboratory tests, liver magnetic resonance imaging (MRI) (2-dimensional [2D] proton density-fat fraction) and NAFLD severity markers were compared between obese group and overweight group. Correlation analyses were performed between serum ferritin and laboratory values including NAFLD severity markers.ResultsIn obese group, serum ferritin, alanine aminotransferase (ALT), total bilirubin, international normalized ratio (INR), MRI 2D proton density-fat fraction, aspartate aminotransferase (AST) to platelet ratio index (APRI) and fibrosis-4 (FIB-4) (an index score calculated from platelet count, ALT, AST and age) were significantly higher than those of overweight group. NAFLD severity markers, APRI and FIB-4, and liver specific important laboratory values, AST, ALT, INR, cholesterol, triglyceride and low density lipoprotein show significant correlation with serum ferritin in NAFLD patients.ConclusionSerum ferritin concentrations could be a candidate of useful severity marker in the pediatric NAFLD patients.

Modification of cellular immune responses in experimental autoimmune hepatitis in mice by maitake (Grifola frondosa)

Immune response to liver-specific lipoprotein (LSP) is involved in the pathogenesis of chronic active hepatitis. Experimental hepatitis could thus be prepared in C57BL/6 mice by injection of liver-specific protein in a syngeneic liver homogenate with Freund's complete adjuvant. In hepatitic mice treated with maitake (Grifola frondosa) fruit bodies, the values of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) values increased temporarily by 2.24–2.79 times and decreased rapidly thereafter. However, in the mice given normal feed, both values increased constantly. Thus, we examined T cell activities both in the exacerbation and remission stages of hepatitis. We suggest that the activation of CD8+ cells is more potentiated than that of CD4+ cells by administration of maitake or the D-Fraction-glucan (β-1,6 glucan having β-1,3 branches), which can enhance immuno-competent cells at the exacerbation stage. However, at the remission stage, marked potentiation of CD8+ cell activity was not observed. These results suggest that depressed suppressor T cell activity is revived by the X-Fraction-glucan (β-1,6 glucan having α-1,4 branched glucan), while the cytotoxic T cell activity, which is activated by the D-Fraction, is restricted, thereby creating a smooth shift from the exacerbation stage to the remission stage.

Experimental Hepatitis in Neonatally Thymectomized Mice: Transfer of Disease and the Role of T Cells

Using neonatally A/J thymectomized mice, we have produced chronic hepatitis by administration of sublethal doses ofPropionibacterium acnesand lipopolysaccharide (LPS). Our goal in this unique model was to evaluate the effector cell population required to generate chronic hepatitis by transferring spleen cells or splenic subpopulations derived from donor thymectomized mice with chronic hepatitis into congenic recipient nonimmunized thymectomized or sham-thymectomized animals. Several key observations were made regarding the ability to induce and to transfer disease. First, an inflammatory liver injury in neonatally thymectomized (NTx) mice was readily generated using sublethal doses ofP. acnesand LPS. Second, the lesions were persistent and associated with the production of autoantibodies to liver-specific lipoprotein and anti-nuclear antibodies. Third, these features were not found in comparably injected nonthymectomized control A/J mice. Fourth, the same liver injury was transferred to neonatally thymectomized but otherwise naive mice by the transfer of donor spleen cells from affected mice previously induced to develop experimental hepatitis. Fifth, the transfer of this liver injury could not be achieved using T-cell-depleted spleen cells. Deletion of CD4+T cells or CD8+T cells by sensitized spleen cells resulted in suppression of the transferred liver injury. In contrast, transfer of nylon wool adherent splenic T cells induced severe hepatitis. These data suggest that the chronic liver injury induced in NTx mice by administration ofP. acnesand LPS involves a breakdown in tolerance accompanied by the appearance of autoantibodies and that nylon wool adherent CD4+and CD8+T cells play important roles in the modulation of liver injury.

T-cell-directed hepatocyte damage in autoimmune chronic active hepatitis

To investigate the function of activated T lymphocytes in autoimmune chronic active hepatitis, 7 of 15 T-cell clones from the peripheral blood of 8 patients were studied. These clones showed specificity for liver-membrane antigen with proliferation when stimulated by rabbit liver cell membranes. 6 of these clones reacted with liver-specific lipoprotein complex, and 1 clone (and 3 subclones) responded to the asialoglycoprotein receptor (ASGPR), both known targets of immune attack in autoimmune chronic active hepatitis. 2 of these clones stimulated autologous B lymphocytes to produce liver-membrane-specific autoantibodies and antibody to the ASG PR. These results suggest that liver-membrane-specific activated T lymphocytes in peripheral blood may be important in the autoimmune attack of chronic active hepatitis.

Production of murine monoclonal antibodies against a liver-specific, species cross-reactive antigen in the liver-specific lipoprotein (LSP) preparation

Previous attempts in several laboratories have failed to produce murine monoclonal antibodies (MAbs) against liver-specific, species-cross-reactive, cell surface-expressed antigens in the normal liver preparation known as liver-specific membrane lipoprotein (LSP). In the present study, BALB/c mice were pretreated with a single dose of cyclophosphamide (20 mg/kg), hyperimmunized with human LSP and hybridomas produced by fusion of spleen cells from these mice with murine myeloma (P3-NS1-Ag4-1) cells. To bias selection in favour of MAbs reacting with species cross-reactive epitopes, hybridoma supernatants were screened by ELISA against rabbit LSP. From 70 stable hybridomas, four MAbs were obtained that react with rabbit LSP. One is an IgM antibody and the other three are of IgG2a class. All four react with the hepatic asialo-glycoprotein receptor (HL), a liver-specific, species-cross-reactive component that is normally expressed on the surfaces of hepatocytes. Using a rapid screening technique (an ‘additive’ ELISA), preliminary evidence was obtained indicating that these four MAbs between them recognise three different epitopes on the HL molecule. The results suggest that this is a viable approach for the production of MAbs against autoantigens to which autoreactivity may normally be suppressed.

57. A NEW METHOD FOR DETECTING ANTI LIVER CELL MEMBRANE ANTIBODY USING A HUMAN HEPATOMA CELL LINE

A simple technique for detection of serum autoantibodies that bind to liver membrane antigens on the surface of liver cells has been developed using Alexander cells[PLC/PRF/5]. Alexander cells were incubated with test serum(1:160), washed then adherent antibodies were detected by incubating with 125I-proCein A. Specific binding to cell surface was calculated as the ratio between the counts with patient's serum divided by 2SD above the mean count obtained with serum from controls. The ratio was significantly higher in those with active autoimmune chronic active hepatitis (aCAH)[median 4.5, range 1.1-23.1] when compared to inactive cases [1.5, 0.3-4.1; p<0.01], to alpha-1 antitrypsin deficiency [1.2, 0.3-2.3; p<0.01], and to Wilson's disease[1.7, 0.3-2.9;p<0.01]. The ratio in primary sclerosing cholangitis was similar to active aCAH[4.2, 0.8-6.7]which would support recent evidence implicating autoimmune mechanisms in PSC. In 11 aCAH cases tested for anti-LSP [liver specific lipoprotein] antibody a positive correlation was found between Alexander cellbinding assay & anti-LSP titres( r= 0.56 p<0.02). In 4 aCAH tested @ diagnosis [4.8,3-23.1] the ratio fell after effective immunosuppresive therapy [0.9, 0.6-4.5]. Alexander cell binding assay provides a simple, rapid & sensitive technique to detect specific antibody to liver cell membrane which may help in the management of autoimmune liver disease.

Detection of the cholestatic factor in the liver tissue of patients with acute intrahepatic cholestasis.

A novel lymphokine, which we have designated as cholestatic factor (CF), was produced from peripheral blood lymphocytes of patients with drug-induced allergic intrahepatic cholestasis by stimulation with a causative drug in the presence of the liver soluble fraction containing liver-specific lipoprotein (LSP). Marked reductions in bile flow and bile acid excretion were induced in rats by injecting CF through a mesenteric vein. In order to confirm the presence of CF in the liver tissue of patients, we attempted to detect this lymphokine by using the enzyme-labelled antibody method. As a result, CF was found in the liver tissue of eleven out of thirty-eight patients with acute intrahepatic cholestasis including one with hepatitis A type, one with hepatitis B type, two with hepatitis non-A non-B type, five with drug-induced allergic hepatitis, one with alcoholic hepatitis and one with lupoid hepatitis. In contrast, CF was undetectable in the liver tissue of patients without intrahepatic cholestasis. These results may additionally support our assumption that CF plays an important role in the induction of intrahepatic cholestasis in various liver diseases.

Clinical evaluation of autoantibodies to liver cell membrane specific antigen, liver specific lipoprotein, and Tamm-Horsfall glycoprotein in autoimmune chronic active hepatitis.

An enzyme-linked immunosorbent assay was developed to detect circulating autoantibodies to three liver cell membrane surface antigens, i.e., liver cell membrane specific antigen (LCM), liver specific lipoprotein (LSP), and Tamm-Horsfall glycoprotein (THGP). In autoimmune chronic active hepatitis (autoimmune CAH), the positive rate and mean titer (normal range, less than 5.5 units) for anti-LCM were 100% and 13.5 units before corticosteroid treatment and 100% and 9.9 units during the treatment. The corresponding values for anti-LSP were 84% and 11.8 units, and 81% and 8.9 units, and those for anti-THGP were 84% and 12.3 units, and 81% and 7.9 units. In an autoimmune CAH patient, elevation of the plasma levels of autoantibodies during the treatment apparently preceded the elevation of alanine aminotransferase (ALT). However, the ALT elevation induced by transcatheter arterial embolization was not associated with the elevation of these autoantibodies in an autoimmune CAH patient with hepatocellular carcinoma. In primary biliary cirrhosis, drug-induced hepatitis, and non-hepatic immunological disorders, the production of the three autoantibodies did not directly correlate with liver cell damage. These findings suggest that the elevation of autoantibodies against LCM, LSP, and THGP can be a useful guide for the prednisolone treatment of autoimmune CAH.

Antibody to liver membrane antigens in chronic active hepatitis. IV. Exclusion of specific reactivity to polypeptides and glycolipids by immunoblotting.

The reactivity of sera was examined in patients with autoimmune chronic active hepatitis and other liver diseases by immunoblotting. Polypeptides and glycolipids of liver plasma membrane, liver-specific lipoprotein and kidney membrane were separated and probed with sera from patients and from a rabbit immunized with mouse liver plasma membrane. Chronic active hepatitis sera reacted with a number of polypeptides in the liver plasma membrane preparations; similar but weaker reactivity was observed with sera from patients with other diseases and in some healthy subjects. Chronic active hepatitis sera did not react with glycolipids from liver plasma membrane. The immune rabbit serum reacted with two polypeptides of 180 kd present in liver plasma membrane but absent from kidney membrane, with two polypeptides of 50 kd which were nonliver-specific but species-specific, and with three major glycolipid components of liver plasma membrane: this reactivity thus differed markedly from that of the chronic active hepatitis sera. In studies using dot-blotting, it was found that solubilization of liver plasma membrane in detergents resulted in a marked reduction of the reactivity to liver plasma membrane of chronic active hepatitis sera, but little change in the reactivity of the chronic active hepatitis and other sera with liver-specific lipoprotein by immunoblotting indicated that liver-specific lipoprotein consisted of constituents of liver plasma membrane together with intracellular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a liver cell membrane specific antigen.

Antibodies against human liver cell membranes were raised in rabbits by immunizing 105,000 g precipitate of normal human liver homogenates (insoluble fraction). After absorption with non-liver antigens such as human spleen, kidney, lung homogenates and serum, the anti-liver cell membrane specific antigen (LCM) IgG fraction was prepared. The antibody reacted with both 105,000 g supernatant of the liver homogenate (soluble fraction) and Triton X-100 extract of the insoluble fraction, and with the surface of Chang liver cells. Using affinity columns to conjugate this antibody and other antibodies against non-liver antigens, LCM was prepared from the soluble fraction composed of 70K, 59K, 49K, and 31K proteins. These proteins of LCM were included in liver specific lipoprotein (LSP). The number of LCM proteins was limited to 4, although that of LSP was 8 or more. Furthermore, circulating antibodies against "LCM" were detected in patients with chronic liver diseases type B and autoimmune hepatitis by an 125I-LCM-IgG-Protein A method. Therefore, LCM may be more useful than LSP to study the immunological responses involved in liver diseases.

Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to study (i) binding of rabbit antibodies (raised against litter mate liver plasma membrane fraction) to the immunizing membrane fraction, and (ii) binding of human antibodies to liver membrane fractions and to liver-specific lipoprotein (a liver membrane-derived antigen complex). When assays were conducted using the non-ionic detergent Tween 20 as blocking agent, high non-specific binding was encountered. With the low titre rabbit antisera high binding of non-immune test antibody and of second antibody (anti-rabbit IgG) to the immunogen, and also directly to the solid phase, was found. This was abolished by replacement of Tween 20 in the antibody diluent buffers by a non-reactive protein, casein proving to be a more effective blocking agent than either bovine serum albumin or gelatin. With human sera, high binding of human IgG to the solid phase was noted. This too was blocked by casein, but only when the anti-microbial agent Thimerosal was included in the casein buffer, and when Tween 20 in the wash buffer was replaced by casein-Thimerosal so that the solid phase was exposed to casein before incubation with the test serum. The casein buffers described may prove of general value in solid-phase assays where high non-specific binding is encountered.

Antibody to liver-specific lipoprotein in acute and chronic liver diseases. Its quantitative assay with monoclonal antibody, but without the use of liver-specific lipoprotein.

A double-antibody radioimmunoassay was developed to detect antibody to human liver-specific membrane lipoprotein (anti-LSP antibody) in patients' serum. Anti-human LSP monoclonal antibody was labeled with 125I and anti-anti-LSP antiserum raised in rabbits was used as the first antibody in the assay. Anti-LSP antibody level was quantitatively measured and the assay was shown to be specific. Anti-LSP antibody was found in 5/8 patients with type B acute viral hepatitis (AVH), 3/7 patients with type A AVH, 1/6 patients with non-A, non-B AVH, 10/17 patients with chronic active hepatitis (CAH), 6/16 patients with chronic persistent hepatitis, 13/16 patients with active cirrhosis of the liver and 7/19 patients with primary nonhepatic autoimmune diseases such as glomerulonephritis, systemic lupus erythematosus and rheumatoid arthritis. The mean levels of anti-LSP were increased in patients with cirrhosis of the liver (p less than 0.01), CAH (p less than 0.05) and AVH (p less than 0.05) when compared with that of normal individuals. However, the frequencies of anti-LSP did not depend on HBsAg status. The data showed that anti-LSP antibody can be detected without the use of LSP preparation although it is also found in patients with primary nonhepatic autoimmune diseases.

Analysis of the antigenic composition of liver specific lipoprotein using murine monoclonal antibodies.

Eight monoclonal antibodies have been raised to antigenic determinants within the liver specific lipoprotein complex. Five of these determinants were species and the others non-species specific. No liver specific determinants were identified. Liver specific lipoprotein antigens recognised by the eight monoclonal antibodies were located either on the hepatocyte membrane and/or along the sinusoidal lining wall or in the cytoplasm of liver parenchymal cells. All antibodies but one reacted with the cell membrane of viable human hepatocytes. The molecular weights of four liver specific lipoprotein-determinants were determined by immunoprecipitation. These ranged from 22 000 to 164 000 daltons.

Murine monoclonal antibodies against "liver specific lipoprotein" (LSP) defining three antigenic sites which differ in tissue- and species-distribution and subcellular location.

The tissue and species cross-reactivity of three monoclonal antibodies against human liver-specific lipoprotein (LSP), as well as the subcellular locations of the respective target antigens, has been investigated using an enzyme-linked immunosorbent assay (ELISA). Antibody D6 was widely tissue cross-reactive and bound to human and rabbit but not rat, mouse or guinea pig tissues. This antibody bound to a particulate antigen localized in the microsomal fraction of rabbit liver, and distinct from enzyme markers for plasma membrane (5'-nucleotidase) and endoplasmic reticulum (glucose-6-phosphatase) in its sedimentation properties on sucrose density gradients. Antibody A9/63 bound to human liver and pancreas, but not kidney, spleen, adipose tissue, skeletal muscle, small intestine or heart, and also bound only to human and rabbit tissues. This antibody bound to a particulate antigen in pancreas, but a soluble antigen in liver. Antibody B20 bound to all tissues from all species tested, with the exception of guinea pig, and bound to particulate antigens in adipose tissue and pancreas but soluble antigens in other tissues (including liver). In addition to emphasizing the immunochemical complexity of LSP, these experiments demonstrate the suitability of monoclonal antibodies for analysis of its constituent antigens.

Possible participation of cholestatic factor in the pathogenesis of intrahepatic cholestasis in alcoholic liver disease.

To elucidate the immunopathogenesis of intrahepatic cholestasis in alcoholic liver disease, the authors investigated the possible participation of a cholestatic factor. The peripheral lymphocytes from patients with intrahepatic cholestasis in alcoholic liver disease were stimulated with ethanol and liver specific lipoprotein in vitro and the culture supernatant was fractionated by gel filtration, using a Sephadex G-75 column. When a definitive fraction (fraction 4) was injected into the mesenteric vein of rats, a marked reduction in bile flow was observed. Similar results were obtained when the blood serum of patients was fractionated in an identical manner and the same fraction was injected into rats via the mesenteric vein. Histologically, dilated bile canaliculi with diminution of microvilli and increased vesicles around the dilated canaliculi were observed by electron microscopy after injection of the active fraction into rats. These results strongly suggest that not only the sensitized lymphocytes produce the cholestatic factor which caused the intrahepatic cholestasis by specific stimulation, but also this factor is involved significantly in the pathogenesis of intrahepatic cholestasis which is observed in patients with intrahepatic cholestasis in alcoholic liver disease.

Antibodies to Liver-Specific Lipoprotein in Children with Chronic Liver Disease Due to “Autoimmune” Chronic Active Hepatitis, Cystic Fibrosis, and α1-Antitrypsin Deficiency

Antibodies to a liver-specific lipoprotein complex (LSP) were determined by radioimmunoassay in the sera of 65 children with possible chronic liver disease. Thirteen had "autoimmune" chronic active hepatitis (CAH), 21 alpha 1-antitrypsin deficiency PiZ (alpha 1-ATD), all having significant liver disease, while 10 of 31 children with cystic fibrosis (CF) had abnormal biochemical tests of liver function, six having cirrhosis. Sera from 12 (92%) of 13 untreated CAH patients contained detectable anti-LSP antibodies, the highest titres occurring in those with anti-liver/kidney microsomal or smooth muscle antibodies. Titre of anti-LSP antibodies correlated with piecemeal necrosis of periportal hepatocytes in liver biopsies, but not with standard biochemical tests of liver function or serum immunoglobulin concentrations. The incidence of LSP antibodies fell as liver damage improved with immunosuppressant therapy, being positive in 18 of 26 sera from children in whom the transaminases were still above normal, but positive in only two of 20 in whom transaminase values were within the normal range (chi 2 = 16, p less than 0.001). Sera from two of 31 patients (6%) with CF contained anti-LSP antibodies as did sera from six of 21 patients with alpha 1-ATD. Antibody concentrations were lower than in CAH, and in alpha 1-ATD the presence of anti-LSP could not be correlated with the presence or absence of piecemeal necrosis on liver biopsy. Our data suggest that autoimmune mechanisms involving antibody to hepatocyte membrane components have a role in the pathogenesis of chronic liver disease in autoimmune CAH in children as in adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and preliminary characterization of monoclonal antibodies to human liver-specific lipoprotein (LSP).

Monoclonal antibodies (mc/anti-LSP) have been prepared by polyethylene glycol fusion of P3/NS1/Ag4-1 myeloma cells with spleen cells from Balb/c mice hyperimmunized with human liver-specific membrane lipoprotein (LSP). Ten hybridomas, cloned by limiting dilution, produced mc/anti-LSP reacting (by ELISA) with human LSP but not with normal human plasma proteins nor with a variety of other proteins likely to co-purify with LSP. Three of these ( A15 /7, A9/63 and B20 ), producing high-titre IgG1 mc/anti-LSP, were biosynthetically radiolabelled and used as index antibodies. By competitive inhibition of binding of the index antibodies to LSP in an immunoradiometric assay, the ten hybridoma products were classified into four distinct groups according to their specificities for different epitopes in LSP. None of the index antibodies reacted, on ELISA, with glutaraldehyde-fixed PLC/PRF/5, Chang, Daudi or HSB-2 cell lines nor with human peripheral blood leucocytes. However, A15 /27 (but not A9/63 or B20 ) reacted with saponin-permeabilized PLC/PRF/5 and Chang cells and also with rabbit LSP. The results emphasize the polyantigenic nature of LSP and indicate that at least one of the mc/anti-LSP ( A15 /27) recognises a species cross-reactive antigen that is present in liver-derived cell lines.

Organ-specificity and clinical significance of antibody to insoluble liver cell mebrane antigen (anti-LM) in sera from patients with chronic active liver diseases

The specificity of insoluble liver cell membarne antigen (LMAg) different from liver specific lipoprotein (LSP), was investigated by immunodiffusion using 2% polyethylene glycol (PEG-ID), immunofluorescence technique and enzyme-linked immunosorbent assay (ELISA). Serum anti-LM against acetone-fixed rat liver was detected in 9 of 13 patients with anti-HBs-positive chronic liver diseases (CLD) and 10 of 25 healthy persons with positive anti-HBs by immunofluorescence technique. Results of ELISA indicated that sera of 21 patients with lupoid hepatitis from Intractable Liver Diseases Committee, the Ministry of Health & Welfare, Japan and 25 patients with HBsAg-negative CLD had high levels of anti-LM. In immunohistological demonstrations of LMAg and LSP, the anti-LM prepared from an anti-HBs-positive patient with chronic active hepatitis bound to each of the human and rat acetone-fixed liver cell membranes, but not bind to any of human and rat kidney tissues. The absorbed rabbit anti-rat LM also bound to liver cell membranes, but the absorbed anti-rat LSP lacked organ-specificity to acetone-fixed liver tissues. Results of PEG-ID indicated that the absorbed rabbit antirat LM had precipitated to two organ-specific components of rat liver homogenate, and one of the two was identified as the one of the absorbed anti-LSP.In conclusion, it is indicated that the appearance of serum anti-LM is associated with a host immune response against the surface antigen components on hepatitis B virus (HBV).

Lipid peroxide formation in isolated hepatocytes by cytotoxic factors produced from lymphokine-activated macrophages.

When peripheral blood lymphocytes from patients with chronic active hepatitis were stimulated with liver specific lipoprotein (LSP), considerably higher frequencies of lymphocyte transformation and MIF production were induced. Peritoneal macrophages from guinea pigs were activated by lymphokine-containing lymphocyte culture supernatant and produced a cytotoxic (or cytostatic) factor acting on isolated hepatocytes in culture. The cytotoxic (or cytostatic) factor, which was fractionated by Sephadex G-75 column gel filtration followed by DEAE-cellulose column chromatography, had cytotoxic effect on isolated liver cells and produced a significant amount of lipid peroxide. These results suggested the possibility that the cytotoxic effects may be caused at least partially by the lipid peroxide formation.

The plasma membrane origin of liver-specific protein (LSP).

Liver-specific lipoprotein (LSP) is a potentially important target antigen for autoimmune liver cell injury, but the complexity of LSP "the preparation" has hindered investigation of anti-LSP immune responses. In the present study, rabbit LSP and isolated liver plasma membranes (LPM) were compared using electron microscopy and marker enzyme analysis to determine the subcellular site of origin of LSP. These results suggest that LSP "the preparation" is highly enriched with plasma membrane.