Demonstration of alpha1-antitrypsin in paraffin sections of hepatoma and cirrhosis

Summary Alpha₁-antitrypsin has been examined in formalin-fixed, paraffin-embedded liver specimens from Greek patients with cirrhosis (35 cases) and hepatoma (55 cases) by peroxidase-antiperoxidase (PAP) method. Ring-like AAT globules were found in the non-neoplastic cells in 12% of the cases of hepatoma and in 11% of the cases of cirrhosis. Atypical globules were seen in neoplastic cells in 5.4% of the cases of hepatoma and in 17% of the cases of liver cirrhosis. A diffuse fine granular pattern of AAT distribution was present in 31% of the cases of hepatoma in the neoplastic cells and in 27% of those in the nonneoplastic cells. The relatively high incidence of ring-like AAT-globules, and of atypical globules in cases of hepatoma and cirrhosis is not in agreement with the extremely low gene frequency of Z allele in a Greek population of patients with cirrhosis and hepatoma. Thus, there is some doubt whether AAT-globules in the liver represent a histopathologic marker of genetically determined AAT deficiency. A relationship between AAT deposits and the degree of differentiation of hepatoma was noted in this series. AAT-positive cells were found in 55% of moderately differentiated, in 29% of highly differentiated and in 20% of poorly differentiated hepatomas.     

Adult alpha1-antitrypsin deficiency.

Three adults with alpha 1-antitrypsin deficiency are described. In two of the cases the deficiency was genetically determined (cases 1 and 2), and each demonstrated unusual features of the disease. The liver in case 1 (homozygous) showed cholangiolar hyperplasia which has been recorded only once before. Case 2 (heterozygous) had emphysema and cirrhosis, a combination not previously documented in a heterozygote, in addition to malabsorption. Case 3 represents a case of spurious alpha 1-antitrypsin deficiency with cirrhosis included to emphasize the diagnostic improtance of phenotyping in such cases.     

Galactosamine-induced alpha 1-antitrypsin deficiency in rats. Alterations in plasma glycoproteins and alpha 1-antitrypsin carbohydrate composition.

Administration of D-galactosamine (GalNH2) is known to produce alterations in plasma glycoprotein levels, including alpha 1-antitrypsin. The authors have studied the effects of GalNH2 on circulating protein bound carbohydrates and on the plasma concentrations of two alpha 1-antiproteases, transferrin, IgG, and albumin in rats. The alpha 1-antiproteases from GalNH2-treated rats were isolated and their molecular weight, isoelectric point, and carbohydrate composition compared with those of control rat alpha 1-antiproteases. Total plasma protein, albumin, and transferrin levels in the GalNH2-treated rats do not differ significantly from those of control rats. Plasma protein-bound carbohydrate is decreased significantly in the experimental animals, compared with controls: sialic acid decreased 60%, neutral sugars decreased 43%, and amino sugars decreased 38%. The concentrations of alpha 1-antitrypsin (AAT) and a higher molecular weight alpha 1-antiprotease designated AP2 are decreased by 79% and 38%, respectively. AAT isolated from the plasma of GalNH2-treated rats contains 2-3 fewer moles of sialic acid, 3 fewer moles of neutral sugar, and 2 fewer moles of amino sugar per mole of antiprotease than AAT isolated from controls. AP2 from GalNH2-treated rats contains 1 fewer mole each of sialic acid, neutral sugar, and amino sugar per mole of antiprotease than AP2 from controls. These alterations are similar to those seen in humans with genetically determined alpha 1-antiprotease deficiency.     

Characterization of alpha1-antitrypsin in the inclusion bodies from the liver in alpha 1-antitrypsin deficiency.

alpha1-antitrypsin was isolated from periodic acid-Schiff-positive inclusion bodies from the hepatocytes of patients with alpha1-antitrypsin deficiency and further purified to enable more detailed chemical analysis. Amino acid and cyanogen bromide fragmentation studies showed a close similarity between hepatic and serum (PiMM) antitrypsin in contrast to the carbohydrate analysis, which revealed markedly deficient glycosylation of hepatic antitrypsin. A complete lack of sialic acid and a relative deficiency of all other carbohydrate components could fully explain the difference of approximately 6000 daltons in molecular size between the two proteins. The accumulation of hepatic globules is probably related to the physical properties of the defective antitrypsin, which include marked insolubility and tendency toward aggregation. The results strongly suggest an abnormal amino acid sequence in the peptide chain of the deficient antitrypsin. The interference with glycosylation may be related to steric hindrance.     

Isolation and characterization of alpha 1-antitrypsin in PAS-positive hepatic granules from rats with experimental alpha 1-antitrypsin deficiency.

Chronic galactosamine (GalNH2) administration in rats decreases plasma alpha 1-antitrypsin (AAT) levels to 10-50% of control levels and induces the formation of diastase-resistant, PAS-positive granules, which contain AAT in hepatocytes. This report describes the isolation and purification of hepatic granule AAT by three different methods: solubilization with guanidine hydrochloride followed by gel filtration on Bio-gel A5M, extraction with methylamine and 2-chloroethanol, and solubilization with sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. All three methods yield a single protein which precipitates with anti-rat plasma AAT antibody, and which has an apparent molecular weight of 45,000 daltons, in contrast to the molecular weight of plasma AAT, 50,000 daltons. Unlike plasma AAT, granule AAT contains no sialic acid, galactose, or fucose. Moreover, granule AAT contains a reduced amount of N-acetylglucosamine and an increased amount of mannose, compared with plasma AAT. The carbohydrate content of granule AAT varies with the isolation procedure used. Granule AAT is susceptible to cleavage by endoglucosaminidase H, which indicates the presence of high-mannose type oligosaccharides. Comparison of the molecular weight, carbohydrate composition, isoelectric point, and endoglucosaminidase H sensitivity of granule AAT isolated from rats with GalNH2-induced AAT deficiency with granule AAT from PiZ humans extends the list of similarities between experimental GalNH2-induced AAT deficiency in rats by and genetically determined AAT deficiency in humans.     

Genetic testing for alpha1-antitrypsin deficiency.

PURPOSE: The Alpha Coded Testing Study investigated the risks, benefits, and psychological impact of home genetic testing for alpha1-antitrypsin deficiency. METHODS: In the study, 996 adult individuals requested and returned a home-administered, confidential, fingerstick blood test. RESULTS: Individuals highly rated the benefits of establishing a diagnosis (82%), helping family members (86%), and anticipating peace of mind (79%). 78% of 239 current smokers reported a high likelihood of smoking cessation if diagnosed with AATD. After testing, more than 60% indicated that they would share the results with family and physicians but < 30% would share results with insurance companies. CONCLUSIONS: Confidential home testing for genetic disorders requires a comprehensive program of participant support.     

Production of monoclonal antibodies against inactivated alpha 1-antitrypsin. Cross-reactivity with complexed alpha 1-antitrypsin and application in an assay to determine inactivated and complexed alpha 1-antitrypsin in biological fluids

15 different monoclonal antibodies (mcAbs) have been raised against the cleaved (inactive) form of the serpin alpha 1-antitrypsin (AT). In initial experiments these mcAbs were analysed for their ability to bind the native and the cleaved form of this inhibitor: eight of the 15 mcAbs appeared to react predominantly with cleaved AT. Additional experiments with mixtures of purified native AT, AT complexed to neutrophilic elastase and inactivated AT revealed that all mAbs that preferentially reacted with inactivated AT also bound to complexed AT. Using two of the mcAbs against inactivated AT a quantitative and sensitive sandwich-type radioimmunoassay was developed to determine levels of proteolytically inactivated AT in biological fluids. With this assay increased levels of inactivated AT were found in synovial fluid from patients with rheumatoid arthritis corresponding to about 2.4% (range 0.3-11%) of total AT. Approximately 10% of this inactivated AT appeared to consist of AT complexed to neutrophil elastase. The mcAbs described here further illustrate the structural resemblance between the complexed and cleaved forms of AT. In addition, these mcAbs appear to be useful tools for the study of AT in human disease.     

Change in alpha 1-antitrypsin phenotype after orthotopic liver transplant.

The aim of the present study was to determine the interval after orthotopic liver transplant during which the alpha 1-AT phenotype in the serum changes. Eleven patients could be evaluated because the donor and recipient had different alpha 1-AT phenotypes. The results showed that within 1 to 3 days of the transplant donor alpha 1-AT is already present in the serum of the recipient, and subsequently the transplanted liver continued to synthesize alpha 1-AT of the donor phenotype. The result confirmed that liver parenchymal cells are the main site of alpha 1-AT synthesis.     

Laboratory diagnosis of alpha1-antitrypsin deficiency

The laboratory diagnosis of alpha(1)-antitrypsin (AAT) deficiency (AATD) has evolved over the last 40 years since the first cases of the disorder were reported. It is currently performed in specialized centers, and it requires a combination of different biochemical methods: nephelometric AAT concentration, isoelectric focusing, genotyping, and sequencing. The availability of matrices such as the dried blood spot have facilitated the implementation of laboratory analyses for AATD, but they have also challenged laboratories to develop more reliable and reproducible techniques starting from dried blood. In this article, we describe the protocols we have optimized for AATD diagnosis from dried blood spot, in an attempt to hopefully provide useful information for physicians and scientists involved in this diagnostic line. We also describe the diagnostic flowchart for AATD detection that we have developed accordingly.     

Immunofluorescent detection of alpha1-antitrypsin in paraffin embedded liver tissue.

Alpha1-antitrypsin was detected by indirect immunofluorescence in frozen sections of liver biopsies from patients with clinically and biochemically proven alpha1-antitrypsin deficiency. The antigen could also be demonstrated in those liver specimens of the same patients which were fixed in Bouin's fluid and embedded in paraffin. The cellular localization and the brightness of the fluorescence were the same in both frozen and paraffin sections. Four additional biopsies from three other patients were selected on the basis of PAS-positive diastase-resistant inclusions reported in the hepatocytes. All these biopsies showed bright fluorescence in the cytoplasm of the liver cells although one of the biopsies was stored for as long as eight years. Specific fluorescence was constantly found in the periportal hepatocytes with varying degrees of positivity. No fluorescence was observed in the six control biopsies from patients with various other liver diseases. These findings prove that paraffin embedded specimens are suitable for immunofluorescence detection of alpha1-antitrypsin and that a retrospective study on old paraffin blocks is possible.     

Pathogenesis of liver disease in alpha 1-antitrypsin deficiency

Alpha-1-antitrypsin deficiency (A1ATD) is one of the most common lethal hereditary disorders. Approximately 5 to 10% of the general population carry an "at risk" gene for the development of liver disease or emphysema of the lung. Patients with A1ATD associated liver disease constitute a histologically and clinically heterogeneous group. The pathogenesis of the disease still remains to be elucidated. Recent experimental evidence produced in the transgenic mouse model is in favour of the engorgement hypothesis considering the deposition of amorphous A1AT in the hepatocytes the prime event causing liver disease. This theory, however, fails to explain the clinical observation of the presence of a number of other factors known to cause chronic liver disease in A1ATD patients. The latter observation is in support of the deficiency theory, which explains the pathogenesis of emphysema of the lung, and would point to A1ATD as a predisposition to acquire chronic disease. The meaning of the increased synthesis of stress proteins in patients with liver disease is still speculative.     

Issues in the diagnosis of alpha 1-antitrypsin deficiency

Alpha 1-antitrypsin deficiency is a relatively common genetic disease that is underrecognized and underdiagnosed. Early diagnosis in the asymptomatic patient helps modify lifestyle choices to reduce the risk of emphysema. In 2003, the American Thoracic Society and the European Respiratory Society issued guidelines to improve standards in diagnosing alpha(1)-antitrypsin deficiency. This review highlights key recommendations for diagnosis of alpha(1)-antitrypsin deficiency, including the different types of diagnostic tests recommended in the guidelines. Options for patient treatment will be discussed.     

Inactivation of urinary kallikrein by alpha1-antitrypsin

Summary Proteolytic activity, with azocasein as substrate in the presence and absence of 0.4 IU kallikrein (Padutin) was measured in the 24 h urine fractions of 100 ambulatory patients with hypertension, proteinuria or haematuria. Urinary protein and alpha₁-antitrypsin concentration have also been assayed. There was an inverse relationship between kallikrein activity and urinary alpha₁-antitrypsin concentration (r=0.84;y=39.2e ^–0.009x). Furthermore, kallikrein activity and 24 h urinary alpha₁-antitrypsin excretion were also inversely correlated (r=0.81;y=886.4e ^–0.011x). Our data suggest an inactivation of renal kallikrein by urinary alpha₁-antitrypsin.This work was supported by the Deutsche Forschungsgemeinschaft (Ho 781/3-2)     

Immunohistochemical localization of alpha 1-antitrypsin in normal mouse liver and pancreas.

Using horseradish peroxidase and fluorescence immunohistochemistry, alpha 1-antitrypsin (alpha 1AT) was demonstrated in normal mouse hepatocytes and pancreatic islet cells. All hepatocytes were positive; 1--3% stained intensely for alpha 1AT. These were located mainly in the periportal area as well as randomly distributed, both singly and in clusters, throughout the liver lobule. Nonparenchymal liver cells were negative for alpha 1AT. The type of hepatocyte cytoplasmic staining appears to after during ontogeny, changing from a localized granular to a diffuse pattern. The use of immunohistochemistry to demonstrate alpha 1AT in normal mouse liver allows us to examine the acute phase response at a cellular level.