Sensitisation to Mallory bodies (alcoholic hyalin) in alcoholic hepatitis.

Using a leucocyte migration inhibition test sensitisation to Mallory bodies (alcoholic hyalin) was found in a statistically significant 41% of 17 patients with alcoholic hepatitis. Patients with alcohol-induced fatty liver and cirrhosis did not demonstrate sensitisation. Mallory bodies are a characteristic feature of alcohol-induced liver damage, and immunological sensitisation to them might lead to liver cell death and cell progression of the hepatitis process.     

Alcoholic hyalin and interstitial filaments as markers of alcoholic damage of internal organs

Biopsies of the liver, stomach, pancreas, small salivary glands, lung of patients with chronic alcoholism were studied morphologically including ultrastructural analysis. The formation of fibrillar alcoholic hyalin in hepatocytes is the most characteristic ultrastructural feature of the liver alcoholic damage. Accumulation of intermediate filaments in the epithelial cells of the stomach, pancreas, small salivary glands, stomach macrophages is a characteristic sign of the alcoholic damage to these organs. This disturbance of the structural organization of the cytoskeleton seems to reflect secretory insufficiency of the epithelium and failure of macrophagal function.     

Pulmonary cytoplasmic hyalin resembling Mallory's alcoholic hyalin in the liver

Sixty-three consecutive autopsy cases of interstitial fibrosis of the lung, 6 cases of organizing pneumonia, 14 of pneumocystis pneumonia, and 20 of acute bacterial pneumonia complicating as a terminal illness listed in our Department of Pathology during a period from 1978 to 1983 were surveyed for Mallory body-like cytoplasmic hyalins in the alveolar cells. We found the hyalins in 10 of 63 cases (15.9%) with interstitial fibrosis of the lung and one of 6 cases with organizing pneumonia. Seven of the former 10 had an associated malignancy; 3 esophageal cancers, 2 lung cancers, and 2 leukemias. Five of the seven patients received an irradiation for treatment of their malignancies, subsequently developed interstitial fibrosis of the lung. Among the remaining 3 of the 10, one showed diffuse interstitial fibrosis associated with rheumatoid arthritis and two had an idiopathic type of diffuse pulmonary fibrosis. There was only one case in which the pulmonary hyalins were found in the absence of extensive interstitial fibrosis within small organizing foci of peribronchial and subpleural location. Pulmonary hyalins showed the same conventional staining properties and ultrastructural features as Mallory's alcoholic hyalins found in the liver, but did not reveal a simultaneous association with the hepatic hyalins. Pulmonary hyalins frequently stained positively with monoclonal anti-cytokeratin antibodies, more strongly at their periphery. Pulmonary hyalins were considered to be a non-specific reaction of alveolar cells to injuries, mostly in association with the pulmonary fibrosis of any etiology but not the hepatic hyalins.     

Centrosomes, microtubules, and microfilaments in the reendothelialization and remodeling of double-sided in vitro wounds.

The maintenance of endothelial monolayer integrity is an important function of aortic endothelial cells. Our study was designed to test two hypotheses related to the repair of the wounded endothelial monolayer. First, that the reappearance of the highly ordered cobblestone monolayer after wound closure is associated with specific sequential changes in the cytoskeletal system. Second, that there are different patterns of reendothelialization depending on whether microfilaments or microtubules are disrupted. One and a half millimeter wide wounds were created down the middle of confluent endothelial monolayers so that there were two wound edges facing each other. During the initiation of repair, the centrosomes of the cells on both sides of the wound reorientated to the front of the cell. The dense peripheral band of actin microfilaments disappeared, the cells elongated and migrated as a uniform sheet with wound closure occurring within 60 hours. The rate of closure remained constant until the migrating fronts met. The cytoskeletal changes observed and the rate of closure were similar to those we reported in a single edge wound. At closure, however, there was a transient piling up of cells which disappeared after 24 to 36 hours. Within 36 to 48 hours after closure, the centrosomes became randomly distributed around the nucleus. By 40 to 48 hours, the dense peripheral band started to reappear and the cells returned to a cobblestone appearance 72 to 96 hours after closure. Thus, the remodeling of the confluent monolayer after wound closure occurs in association with a specific series of cytoskeletal changes. When the microfilaments were disrupted with cytochalasin B, cell migration still occurred but it took up to four times longer for closure. These cells were initially flatter than normal and contained only a few microfilament fibers; however, the microtubule system was intact and centrosome reorientation occurred, but at a slower rate. However, when the microtubules were disrupted with colchicine, either at the onset of wounding or during repair, neither centrosome reorientation nor cell migration occurred. Thus wound closure of small-sized wounds require the presence of intact microtubules, whereas the additional presence of microfilaments results in a more rapid and efficient system of reendothelialzation.     

Mallory bodies. Horseradish peroxidase: specific cytochemical and biochemical marker for alcoholic hyalin.

Horseradish peroxidase (HRP), a glycoprotein enzyme, bound specifically to Mallory bodies (MBs) in cryostat sections of autopsy liver and liver biopsies. In contrast, HRP did not bind to cryostat sections of normal liver. The specificity of HRP binding was also observed using light and electron microscopy in autopsy liver-derived subcell fractions prepared by the MB isolation procedure. In order to quantitate HRP binding, a solid phase colorimetric assay was developed. This assay involves immobilizing purified MBs or homogenized tissue fractions in glass tubes, incubating with HRP, and measuring the enzymatic activity of bound HRP. A linear relationship between MB concentration and HRP binding was observed. The assay was capable of detecting as little as 1 microgram of MB protein. The specificity of HRP binding was also investigated using the solid phase assay. The specific activity (HRP bound per milligram of protein) of purified MBs was 10 to 15 times that of a glass wool-filtered liver homogenate suggesting that the solid phase assay may be of use in monitoring the purification of MBs. HRP did not bind to normal liver homogenate even when large loads were assayed. The results of this study indicate that HRP binding, employed cytochemically, represents a rapid and facile procedure for ascertaining the presence of MBs in tissue. In some cases, those structures may not be easily visualized by conventional staining procedures. Furthermore, quantitation of MBs in tissue may be possible by using a solid phase enzyme-linked assay.     

Possible roles for microtubules and microfilaments in ADH action on toad urinary bladder.

Intramembranous particle aggregates in the luminal membrane of toad bladder granular cells after vasopressin stimulation have been found to correlate closely and specifically with induced alterations of water permeability. Roles for microtubules and microfilaments in mediating the latter response have been proposed on the basis of studies involving colchicine and cytochalasin B, respectively. In the present investigation the effects of these agents on both initiating and sustaining vasopressin-induced osmotic water flow and the particle aggregation phenomenon were studied. The results indicate that during initiation the aggregation and water flow responses to vasopressin are each colchicine- and cytochalasin B-sensitive and that these sensitivities can be wholly additive. However, after full vasopressin stimulation is established, the same responses demonstrate sensitivity only to cytochalasin B, not to colchicine. The findings, therefore, suggest that microtubules and microfilaments may be independently necessary for the initiation of the aggregation and water flow responses to vasopressin, and that microfilaments, but not microtubules, are required for their maintenance.     

Alcoholic hepatitis in females

In the period 1970-1984 alcoholic hepatitis was diagnosed by liver biopsy in 52 females. Thirty-six patients with cirrhosis were generally in a worse clinical and biochemical state than those without cirrhosis. Biochemical tests for liver function showed significant improvement from admission to the time of liver biopsy. At follow-up liver function tests were generally better in patients who had stopped drinking alcohol compared to those who continued to do so. The 5-year survival rate was 82% for females without cirrhosis, and 45% for those with cirrhosis (p less than 0.03). Considering the sex-related differences in alcohol abuse in the general population we found no evidence of increased susceptibility to the hepatotoxic effect of alcohol in females.     

Morphological variants of alcoholic hyalin and possible ways of its evolution]

Possible ways of alcohol hyalin evolution were studied by repeated electron microscopic examinations of the livers of 2 patients with acute alcoholic hepatitis Four types of ultrastructure of alcoholic hyalin were found: parallely oriented fibrillae, randomly oriented fibrillae, fine granular and large granular (spotlike) substance. Examinations of the material from repeated biopsies showed fibrillar hyalin to be "young" and to turn into "old" hyalin of granular structure. The evolution of alcoholic hyalin from fibrillar to granular is accompanied by hepatocyte necrosis and leukotaxic effect.     

Liver injury with perivenular fibrosis and alcoholic hyalin after pancreatoduodenectomy for pancreatic carcinoma

We describe a patient who developed progressive hepatic failure one year after pancreatoduodenectomy for pancreatic carcinoma and died of gastrointestinal bleeding. He suffered from progressive weight loss after surgery, even though obstruction or stenosis of the gastrointestinal tract was excluded. At autopsy, the liver showed extensive perivenular fibrosis associated with variable loss of hepatocytes, perisinusoidal fibrosis, alcoholic hyalin and a lack of parenchymal regenerative activity, all of which closely resembled severe alcoholic liver disease. Stricture of both the main pancreatic duct and the pancreaticojejunostomy with almost complete loss of exocrine acini was also found, and the recurrent tumor was seen to have caused portal venous obstruction and hepatic arterial stenosis. A combination of these nutritionally unfavorable circumstances and prolonged ischemia appeared to have been responsible for the liver injury in this non-alcoholic patient.     

Complement-mediated release of histamine from human basophils. III. Possible regulatory role of microtubules and microfilaments.

The release of histamine by normal human leukocytes (basophils) following in vitro challenge with activated complement (zymosan-treated serum) was previously reported. In this study, the effects of various pharmacologic agents on this release mechanism were compared with allergen-induced release of histamine. Colchicine and vinblastine antagonize the polymerization of tubulin to form microtubules, and both agents inhibited complement-and allergen-triggered release of histamine from basophils. Finally, treatment with cytochalasin B, a fungal product known to interfere with microfilament formatin, resulted in enhanced release of histamine from complement-treated basophils but no significant change in the percentage of histamine released from allergen-treated basophils. These findings suggest that microtubules and/or microfilaments are involved in complement-induced secretion of histamine by human basophils.     

Activation of human basophils by staphylococcal protein A. I. The role of cyclic AMP, arachidonic acid metabolites, microtubules and microfilaments

Protein A from Staphylococcus aureus (Staph A) induces histamine secretion from human basophil leucocytes in the concentration range 10(-4) - 10 micrograms/ml. This reaction has great similarities to that of antigen or anti-IgE-induced release. It is characterized by a two stage reaction, requires extracellular calcium and is optimal at 37 degrees C. The rate of release is similar to that of IgE-mediated reactions. Histamine release induced by Staph A is inhibited by metabolic inhibitors, drugs which increase intracellular cyclic AMP levels, inhibitors of lipoxygenase pathways and a phospholipase A2 inhibitor. D2O and cytochalasin B which affect microtubules and microfilaments respectively, enhance histamine release induced by Staph A. These results suggest that Staph A-induced release is modulated by intracellular cyclic AMP, arachidonic acid metabolites, requires energy and is enhanced by the disruption of microfilaments and stabilization of microtubules.     

An improved fixation procedure for microtubules and microfilaments in cells of the anterior pituitary gland

A fixation procedure for the preservation of microtubules and microfilaments in anterior pituitary cells is presented. Small pieces of tissue were fixed at room temperature in a glutaraldehyde solution. The temperature was gradually reduced to 4–5°C before the tissue was post fixed. Post fixation was carried out using several changes of a mixture of cold glutaraldehyde and osmium tetroxide and then osmium tetroxide alone. The osmolarity of both fixing solutions was 400 milliosmoles. The procedure permits a clear visualization of both microtubules and microfilaments within anterior pituitary cells, particularly in regions where they are not ordinarily seen such as within the Golgi apparatus.     

Lymphocyte sequestration by the liver in alcoholic hepatitis.

The possibility that lymphocytes are sequestered by the liver in patients with alcoholic hepatitis was investigated. Sixteen patients who had liver biopsy specimens taken were studied as follows: The T- and B-lymphocyte populations of the peripheral blood and liver biopsy digest were quantitated. The liver biopsy specimens were studied by light and electron microscopy for lymphocyte-hepatocyte interaction. The data were analyzed by comparing the results of patients with and without the presence of Mallory bodies (MBs) in the liver. When MBs were present, the percentage of T cells was significantly increased in the liver compared with peripheral blood. By electron microscopy, two livers with MBs showed lymphocyte-hepatocyte interaction. The results support the concept that lymphocytes participate in a cell-mediated immune process when MBs are present in the liver.     

Centrioles,microtubules and microfilaments in activated mononuclear and multinucleate macrophages from rat peritoneum: Electron-microscopic and immunofluorescence microscopic studies

In male Wistar rats of the BD I strain, mononuclear macrophages and multinucleate giant cells obtained from the peritoneum 1 day to 5 weeks after implantation of coverslips coated with dermoid cyst sebum, were examined by light microscopy and immunofluorescence microscopy, using antibodies specific for actin and tubulin and also by scanning and transmission electron microscopy. In activated mononuclear macrophages, microtubules radiate, from the centrioles, situated in the perinuclear area, into the cytoplasm and the major cell processes. Microfilaments form a dense meshwork beneath the plasmalemma. When mononuclear macrophages fuse to form multinucleate giant cells, the initially unordered (“Foreign body”) syncytia still reveal the original distribution patterns of centrioles, microtubules and micro-filaments similar to those seen in the individual cells. In the ordered (Langhans) multinucleate giant cell all centrioles are accumulated in a main pluricorpuscular central group. Centrioles are the initiating and organising centres in the formation of microtubules. From the centrioles microtubules extend into the entire cytoplasm of the syncytium as a uniformly organised, stellate, radial system. The centrosphere, which is characteristic for ordered multinucleate giant cells, seems free from micro-filaments, which form a ring-shaped woven network encircling the nuclei. Depolymerisation and inhibition of microtubules upon exposure to colchicine, indicates that both the organisation of the cytoplasm and the cellular movements depend on the undisturbed coordination of centrioles, microtubules and microfilaments. This applies also to the fusion of mononuclear macrophages to form syncytia, the ordering processes within multinucleate giant cells, and the function of ordered giant cells.     

Sclerosing hyaline necrosis in noncirrhotic chronic alcoholic hepatitis.

A group of 18 chronic alcoholic patients who had sclerosing hyaline necrosis in noncirrhotic livers was compared with a group of 12 similar individuals with acute alcoholic hepatitis, but no centrilobular fibrosis. In cases with sclerosing hyaline necrosis, the most characteristic features were portal hypertension with very large, tender livers and unusually high glutamic-oxalacetic transaminase values; these were associated with centrilobular fibrosis and abundant alcoholic hyalin. Three of these patients died within two years and in two of these, early cirrhosis was found at necropsy. In the cases of acute alcoholic hepatitis, hepatomegaly was the most conspicuous finding, and only a single patient died; death here was unrelated to hepatic disease, the liver being unremarkable at necropsy. Patients who had sclerosing hyaline necrosis tended to remain ill for significantly longer periods. These observations, in conjunction with evidence gathered from the literature, seem to suggest that sclerosing hyaline necrosis is an obligatory step in the natural evolution of alcoholic hepatic disease, especially in cases that evolve into cirrhosis.     

Solid-phase radioimmunoassay for detection of alcoholic hyalin antigen (AHAg) and antibody (anti-AH).

Alcoholic hyalin (AH) was isolated and purified from post-mortem livers of patients with alcoholic hepatitis. Antibodies against AH (anti-AH) were raised in guinea-pigs. Their specificity was demonstrated by immunofluorescence and by immunoabsorption. The antisera were positive in immunofluorescence and complement fixation up to serum dilutions of 1:320 and 1:32 respectively. In order to achieve a higher sensitivity a solid-phase radioimmunoassay (SP-RIA) was established for detection of alcoholic hyalin antigen (AHAg) and anti-AH. Anti-AH could thus be measured up to a 10(5) serum dilution. Using a blocking test, solubilized AH at protein concentrations as low as 80 to 160 ng/ml could be detected. With this SP-RIA, neither AHAg nor anti-AH was found in the sera of 32 patients with histologically proven alcoholic hepatitis. The sera were likewise negative by indirect immunofluorescence, complement fixation and immune adherence haemagglutination test.     

"Alcoholic hepatitis" in a hepatic adenoma

A unique hepatic adenoma developed in a 26-year-old woman who had used oral contraceptives for 10 years and Tolinase (tolazamide sulfonylurea) for adult-onset diabetes mellitus for five years. Clinically, radiographically, and grossly, the neoplasm showed the usual features of a hepatic adenoma, but microscopically it strongly resembled alcoholic hepatitis with steatonecrosis and Mallory bodies. The surrounding hepatic parenchyma was entirely normal. On transmission electron microscopy these Mallory bodies appeared to be tangles of intermediate filaments. They stained readily with antibodies to cytokeratin but not with antibodies to epidermal keratin or vimentin, just as in "alcoholic" hyalin.     

Elevated plasma interleukin-6 and increased severity and mortality in alcoholic hepatitis.

Recent studies in alcoholic hepatitis have proposed a role for the cytokine tumour necrosis factor-alpha (TNF-alpha) a mediator of endotoxic shock in sepsis. In this study plasma levels of the closely related cytokine interleukin-6 (IL-6) were assayed in 96 samples from 58 patients with severe alcoholic hepatitis, and 69 patients in control groups (21 normal, 10 alcoholic without liver disease, 10 inactive alcoholic cirrhosis, 18 chronic liver disease, 10 chronic renal failure). Plasma IL-6 levels were markedly elevated in patients with alcoholic hepatitis when compared with all control groups (P less than 0.001). IL-6 levels were higher in patients who died (P = 0.04) and correlated with the features of severe disease including: increased grade of encephalopathy, increased neutrophil count, increased prothrombin ratio, hypotension, increased serum creatinine and increased serum bilirubin. Surprisingly, no correlation was found between levels of plasma IL-6 and plasma TNF-alpha or endotoxin, or the presence of infection; an inverse correlation was found between plasma IL-6 and serum globulins. These findings provide further evidence that the IL-6/TNF cytokine system is activated in severe alcoholic hepatitis and may mediate hepatic or extra-hepatic tissue damage.