Alpha-1-antitrypsin deficiency and hepatocellular carcinoma.

Forty-two cases of hepatocellular carcinoma (HCC) were examined for the presence of the inclusions of alpha-1-antitrypsin (AAT), which indicate a carrier state for the Pi Z gene. These were found in the non-neoplastic liver tissue of two cases of HCC and in one of the 98 control livers, a difference that is not statistically significant. Typical globules of AAT deficiency were not found in HCC cells. One-quarter of HCCs, however, contained cells which showed diffuse cytoplasmic staining for AAT, a pattern seen also in the non-neoplastic livers.     

A case of liver cirrhosis with alpha-1-antitrypsin globules and hepatitis B surface antigen seropositivity

PAS staining, immunohistochemical examination and electron microscopy revealed presence of alpha-1-antitrypsin (AAT) globules in the hepatocytes of a HBsAg and anti-HBc seropositive female patient diseased of liver cirrhosis. The possible causes of cirrhosis are briefly analysed and the diagnostic importance of PAS-positive, amylase-resistant hepatocellular inclusions is discussed. Apart from the case reported, only two of 509 cirrhotic livers of adults, examined either by biopsy or post mortem, demonstrated similar characteristic PAS-positive globules. This indicates that in the population group (135,000 persons) referred for health care to the hospital where the examinations were done, AAT deficiency has played a negligible role in the development of liver cirrhosis in adults.     

Detection of alpha-1-antitrypsin in hepatocytes in acute and chronic hepatitis

Twelve of sixteen consecutive needle biopsies of liver with either acute or chronic hepatitis showed positive immunohistochemical staining for alpha-1-antitrypsin (AAT). Only two of the positive biopsies contained numerous, large periodic acid-Schiff positive, diastase resistant (PAS-D) globules in periportal hepatocytes; both patients were Pi MZ but only one had a low serum AAT concentration. The other 15 patients had normal or elevated serum AAT. The accumulation of AAT in hepatocytes, demonstrated by sensitive immunohistochemical staining, may indicate increased synthesis and/or impaired secretion of AAT occurring in association with various types of hepatitis.     

Use of alpha-1-antitrypsin staining in the diagnosis of nodular regenerative hyperplasia of the liver

Nodular regenerative hyperplasia (NRH) is a disorder characterized by regenerative nodules scattered diffusely throughout the liver without associated fibrosis. It is most often recognized at autopsy when the entire liver is available for inspection. The diagnosis of NRH by needle biopsy is much more subtle. Since alpha-1-antitrypsin (AAT) expression by hepatocytes in a variety of liver diseases has suggested that it may represent a marker for regenerative or damaged hepatocytes, we elected to study the expression of AAT by immunohistochemical staining of paraffin-embedded tissue of biopsy material as a possible marker of this diagnosis. Seventeen biopsies of the liver showing histologic features consistent with NRH were selected and compared with 20 biopsies of the liver without features of NRH. Eight of the NRH cases showed periportal granular AAT staining as opposed to only one of the non-NRH biopsies (P less than .01; Fisher exact test). These results indicate that AAT expression is increased in the regenerating compartment (as opposed to the presumably damaged atrophic portion) of the liver in NRH and suggest that AAT staining may be useful in confirming the biopsy diagnosis of NRH.     

Isoelectric focusing phenotyping and denaturing gradient gel electrophoresis genotyping: a comparison of two methods in detection of alpha-1-antitrypsin variants

Laboratory diagnosis of alpha-1-antitrypsin (AAT) deficiency is routinely performed by phenotyping methods, which include measurement of serum alpha-1-antitrypsin concentration and isoelectric focusing (IEF). Several DNA-based methods are also used for AAT deficiency testing, but they still have not become part of routine diagnostics. The aim of the study was to identify AAT variants using 2 different methods, isoelectric focusing and denaturing gradient gel electrophoresis (DGGE), and to compare obtained results as well as practical application of these 2 methods. The study has encompassed 27 emphysema patients. In all patients, AAT phenotypization was conducted using IEF, whereas genotypization was performed by DGGE. Variations detected by DGGE were characterized by DNA sequencing. Mutations in the AAT gene were detected in 6 patients. Three patients were homozygous for the Z allele, whereas 1 patient was heterozygous. In 2 patients, novel AAT variants, G320R and V321F, were detected. When results obtained by IEF and DGGE were compared, it was observed that IEF results were inconclusive or misinterpreted in 5 cases (18.5%). Both methods proved to be reliable for detection of the Z alleles, whereas discrepancy existed for M4 allele and rare variants. Therefore, the optimal strategy for diagnostics of AAT deficiency should encompass detection of the most common AAT variants by IEF and screening for the less common variants by DGGE in combination with sequencing.     

Occurrence of alpha-1-antitrypsin deficiency in 155 patients with alcoholic liver disease

Liver biopsies from 155 patients with alcoholic liver disease were examined for periodic-acid-Schiff-positive, diastase-resistant (PAS-DR) intracytoplasmic globules in hepatocytes. Seven patients had these PAS-DR globules: each was a heterozygote for a deficiency allele of alpha-1-antitrypsin (AAT), or alpha-1-protease inhibitor, with the PAS-DR globules distributed in a pattern characteristic of this deficiency. One further patient with normal AAT had a few intracytoplasmic PAS-DR globules in occasional hepatocytes. The prevalence of AAT heterozygotes in this series did not differ from that in the reference population. The seven heterozygotes included five of PI (protease inhibitor) type MZ, one of PI type SZ, and one heterozygous for a rare deficiency allele, PI type MMmalton. The M and Mmalton alleles may be difficult to distinguish because they have similar mobilities with isoelectric focusing technics. Therefore, if PAS-DR inclusions are found in the liver of a patient with an apparently normal phenotype, the presence of a defective M variant allele, such as Mmalton, should be considered.     

Alpha-1-antitrypsin globules in liver biopsies

In order to examine the frequency of alpha-1-antitrypsin (AAT) deficiency of phenotype Pi-Z in a consecutive liver biopsy material, PAS/diastase resistent globules with positive immunohistochemical reaction for AAT (AAT globules) were used as a marker of the Pi-Z gene. 34 (4%) of 850 liver biopsies contained AAT globules. More than half of the biopsies with globules had chief histological diagnoses within the groups fibrosis, suspicion of cirrhosis and cirrhosis. Micronodular cirrhosis was significantly more frequent in biopsies with AAT globules. The results support the assumption that AAT deficiency of phenotype Pi-Z as well in homozygous as heterozygous form is associated with development of liver cirrhosis.     

Alpha-1-antitrypsin and copper in the liver

The incidental finding of orcein positive granules, indicating copper associated protein, in alpha-1-antitrypsin (AAT) positive liver biopsies stimulated a histochemical search for evidence of copper and copper-binding protein in a series of 46 liver biopsies with histological evidence of AAT accumulation. Hepatic accumulation of copper and copper-binding protein occurred in all 19 cirrhotics (100%) and in 14 out of 27 non-cirrhotic livers (51.85%). The overall percentage was 71.73%. AAT and copper deposits coexisted in the same periportal hepatocytes. AAT globules showed positive reactivity both to rhodanine and orcein stains. The severity of chronic liver damage correlated with increasing amounts of copper deposition. It is suggested that in AAT storage, not only is the metabolism of this substance disturbed, but also that of proteins involved in copper metabolism and excretion, resulting in copper accumulation within hepatocytes.     

Hereditäre Hämochromatose, Alpha-1-Antitrypsin-Mangel und Morbus Wilson

Primary hemochromatosis, alpha-1-antitrypsin (AAT) deficiency, and Wilson's disease are the most common hereditary causes of unclear hepatopathy. Classical primary hemochromatosis (type I) on the basis of a homozygous mutation of the HFE gene, usually presents in adults with increasing hepatocellular siderosis and chronic progressive necroinflammatory liver disease. Homozygous AAT deficiency type PiZZ becomes manifest in newborns as a giant cell hepatitis or findings similar to bile duct atresia, in adults as chronic hepatitis or "cryptogenic cirrhosis". The heterozygous PiZ mutation can lead to PAS-positive hepatocellular AAT deposits increasing over the life time. Immunohistochemical detection of AAT deposits by specific PiZ antibodies is a highly sensitive and specific supplementary method. Molecular analysis of AAT and HFE genes in paraffin-embedded tissue or blood can confirm the diagnosis and allows the zygosity status to be defined. Wilson's disease has to be considered in children and young adults with unexplained histologic findings of chronic hepatitis or steatohepatitis. Rhodanin staining is the most effective histochemical method to detect free copper deposits, but negative staining results do not exclude Wilson's disease. In cases suspected of Wilson's disease further clinical exploration must be initiated. The diagnosis is based on a combination of clinical and biochemical findings, which can be supplemented by mutation analysis of the ATP7B gene.     

Alpha-1-antitrypsin hepatic cell globules in autotransplanted liver of rats.

The autotransplantation of liver into the subcutaneous tissues in rats and hamsters resulted in the development of periodic acid Schiffs staining positive granules, which were resistant to diastase digestion. These granules were confirmed as alpha-1-antitrypsin by immunohistochemical staining. The appearance of the alpha-1-antitrypsin containing hepatic granules is comparable to those seen in the liver of humans with genetically determined alpha-1-antitrypsin deficiency.     

Simultaneous alpha-1-antitrypsin accumulation in liver and pancreas

Inclusions positive for periodic acid-Schiff, resistant to diastase, and immunoreactive to alpha-1-antitrypsin (AAT) were found in hepatocytes and pancreatic islet cells of a patient with clinical and pathologic features of AAT deficiency. Alpha-1-antitrypsin was detected in all pancreatic islets, and AAT-positive cells were observed in the excretory pancreatic ducts. These findings suggest that the pancreas synthesizes AAT and possibly serves as a "storage" place in AAT deficiency. Intercalated cells in the excretory pancreatic ducts may be an additional source of AAT.     

Alpha-1-antitrypsin hepatic cell globules in autotransplanted liver of rats

The autotransplantation of liver into the subcutaneous tissues in rats and hamsters resulted in the development of periodic acid Schiffs staining positive granules, which were resistant to diastase digestion. These granules were confirmed as alpha-1-antitrypsin by immunohistochemical staining. The appearance of the alpha-1-antitrypsin containing hepatic granules is comparable to those seen in the liver of humans with genetically determined alpha-1-antitrypsin deficiency.     

Cirrhosis and hepatocellular carcinoma in a patient with heterozygous (MZ) alpha-1-antitrypsin deficiency

A patient is described with micronodular cirrhosis, partial (heterozygous, MZ) deficiency of alpha-1-antitrypsin (AAT) and hepatocellular carcinoma. The patient did not drink alcohol and all serological markers of infection with hepatitis B virus were absent. Death was due to intra-peritoneal bleeding from a multifocal liver tumour. Histology revealed multiple intracytoplasmic AAT globules in hepatocytes at the periphery of the cirrhotic nodules. Copper granules, present in the same non-neoplastic liver cells may have resulted from minor cholestasis. Within the neoplastic hepatocytes AAT globules were sparse and copper deposits co-existed with the globular variant of Mallory bodies. The case is presented in support of the postulated association of partial deficiency of AAT, chronic liver disease and hepatic neoplasia.     

Primary lymphomas of the liver. Report of six cases and review of the literature

Six cases of diffuse large cell lymphoma (DLCL) of the liver were studied with immunohistochemistry for common leukocyte antigen (CLA), lysozyme, alpha-1-antitrypsin (AAT), and kappa and lambda light chains on paraffin-embedded tissues. All six cases were positive for CLA. Four of the six cases showed staining for lysozyme and AAT (three focal and one diffuse staining). In three cases, frozen tissue for monoclonal antibodies and glutaraldehyde-fixed tissue for electron microscopic examination were available. Two of these showed B-cell phenotypes with monoclonal antibody studies. Electron microscopic examination on these two B-cell lymphomas showed scant cytoplasm and a paucity of cytoplasmic organelles. The third case did not show definite B- or T-cell surface markers but did show strong Leu-M1 and OKM1 staining. Electron microscopic examination of the tumor cells showed a prominent Golgi apparatus, abundant cytoplasm with numerous cytoplasmic organelles and phagolysosomes. However, DNA hybridization studies on this tumor showed immunoglobulin heavy and kappa light chain gene rearrangements typical of a B-cell lymphoma. All six lymphomas were solitary liver masses without evidence of disease elsewhere. The mean age for the six patients was 56.2 years (four males, two females).     

Analysis of the two common alpha-1-antitrypsin deficiency alleles PiMS and PiMZ as modifiers of Pseudomonas aeruginosa susceptibility in cystic fibrosis

Lung disease is the direct cause of death in more than 90% of cystic fibrosis (CF) patients. Proteinase-antiproteinase imbalances are common in CF and alpha-1-antitrypsin (AAT) deficiency. We investigated the hypothesis that the AAT deficiency alleles PiS and PiZ contribute to pulmonary prognosis in CF. Two hundred and sixty-nine CF patients from Southern Germany were included in this study. The serum concentrations of AAT and C-reactive protein (CRP) were determined by nephelometry, and patients were screened by polymerase chain reaction (PCR) and restriction enzyme digest for the common AAT deficiency alleles PiS and PiZ. The onset of chronic bacterial colonization by Pseudomonas aeruginosa (Pae) was correlated with the AAT phenotypes PiMM, PiMS and PiMZ. Only three out of nine CF patients (33%) diagnosed with either PiMS or PiMZ had developed chronic Pae lung infection earlier in their lives. The remaining six patients showing a PiMS or PiMZ phenotype showed a later onset of chronic Pae lung infection. Our results indicate that PiMS and PiMZ are not associated with worse pulmonary prognosis in CF. These data need to be confirmed in studies with a much larger number of cases.     

Hepatocytic PAS-positive diastase-resistance inclusions in the absence of alpha-1-antitrypsin deficiency--high prevalence in alcoholic cirrhosis

The presence of PAS-positive, diastase-resistant inclusions in the cytoplasm of the hepatocytes is characteristic of alpha-1-antitrypsin deficiency. The purpose of this investigation was to determine whether the presence of these inclusions is a specific feature, permitting the recognition of alpha-1-antitrypsin deficiency in patients with liver disease. We examined the liver specimens from 20 patients suffering from alcoholic cirrhosis with the Pi M phenotype, i.e., in whom alpha-1-antitrypsin deficiency was excluded. In seven of these patients, PAS-positive, diastase-resistant inclusions were seen in the hepatocytes; in two patients, these inclusions contained a material antigenically similar to alpha-1-antitrypsin. These inclusions might represent deposits of glycoproteins poorly excreted by the diseased hepatocytes. It is concluded that, in patients with liver disease, the presence of PAS-positive, diastase-resistant inclusions--even containing alpha-1-antitrypsin--in the cytoplasm of the hepatocytes does not permit the hepatic lesions to be ascribed to alpha-1-antitrypsin deficiency.     

"The lion, the witch and the wardrobe": impact on sibs of individuals with AAT deficiency

Alpha(1)-antitrypsin (AAT) deficiency is a genetic disorder that may cause serious pulmonary or liver impairment in children or adults. Although genetic sequencing of the AAT gene has only been available for 20 years, analysis of the amount and electrophoretic mobility of the AAT protein has allowed clinical phenotyping for more than 40 years. There have been no studies assessing the psychological impact of having a sib affected by AAT deficiency. Twenty-five participants drawn from the Alpha-1 Research Registry completed a questionnaire and semi-structured interview. Respondents were supportive of testing prior to adulthood for AAT status; 18 thought it was a good idea to test a child, three did not know, and four said children should not be tested, primarily citing insurance concerns. Of those 18 who stated it was a good idea, 14 would test at birth. Knowledge of genetics of AAT deficiency was limited; only 44% of respondents understood the inheritance pattern. We recommend: (1) parents and sibs need help in mourning the loss of children with AAT deficiency; young sibs are at risk for trauma and long-term developmental problems. (2) Teams evaluating donors for liver transplantation should be aggressive in ruling out AAT deficiency prior to invasive testing. (3) Testing should be offered to individuals with a family history of AAT deficiency to obtain the health benefits of lifestyle modification and limit the burden of disease discovery in symptomatic relatives. (4) Awareness of liver disease from AAT deficiency should be increased.     

Rapid DNA analysis of alpha 1-antitrypsin deficiency: application of an improved method for amplifying mutated gene sequences

The gene coding for alpha 1-antitrypsin (AAT) is mutated in some individuals to code for a protein, the PiZ variant, associated with alpha 1-antitrypsin serum deficiency. Homozygotes for this defect are at high risk for various liver diseases and for early onset emphysema. Here, we employ an enzymatic amplification procedure for diagnosis of a Z-specific gene mutation in cellular DNA, and we describe a new procedure for selective enhancement of the mutated sequence. We have synthesized 2 oligonucleotides which prime amplification of a 156 bp region of exon V including a single base mutation specific for the PiZ variant. Amplification of 2 to 6 X 10(5)-fold is obtained allowing rapid detection of the Z mutation, using selective oligonucleotide probes, in unseparated genomic DNA of cells from family members homozygous or heterozygous for the defect. New methodology allows enzymatic amplification at elevated temperatures. To take advantage of this, we have developed a method using discriminating oligonucleotides as primers that allows enhanced sensitivity of hybridization. These methods allow diagnosis of the Z form of AAT deficiency within a single day using submicrogram quantities of DNA.     

Phenotypic characterization of hepatic proliferation. Antigenic expression by proliferating epithelial cells in fetal liver, massive hepatic necrosis, and nodular transformation of the liver

Different forms of hepatocellular proliferation are seen in fetal livers, massive hepatic necrosis, and nodular transformation (nodular regenerative hyperplasia) of the liver. In an attempt to characterize the proliferating cells in these conditions, we studied the expression of several antigens by immunohistochemical methods. Alpha-fetoprotein (AFP), alpha 1-antitrypsin (AAT), a hepatocellular export protein, carcinoembryonic antigen (CEA), a marker of bile duct epithelial cells, and hepatitis B virus antigens (HBsAg, HBcAg), were localized by the peroxidase-antiperoxidase method in 11 fetal livers, 10 cases of nodular transformation, and 7 cases of massive hepatic necrosis. AFP was the most prevalent antigen in fetal hepatocytes. Many hyperplastic hepatocytes in nodular transformation contained AAT, but not oncofetal antigens, supporting the differentiated hepatocellular nature of these cells. A similar staining pattern was seen in two-cell-thick plates of hepatocytes in massive hepatic necrosis. In contrast, the ductlike structures at the periphery of necrotic lobules contained both AAT and CEA, suggesting that these cells exhibit features of hepatocytes and bile duct epithelial cells. Therefore, the appropriate term for these regenerating cells appears to be "ductular" or "biliary hepatocytes".     

In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA

alpha-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI(*)Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI(*)ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.