Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

BACKGROUND AND AIMS: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. METHODS: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA). RESULTS: Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. CONCLUSIONS: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

Fibrous obliterative lesions of veins contribute to progressive fibrosis in chronic liver allograft rejection.

Fibrosis in liver allografts undergoing chronic rejection (CR) is variable and poorly understood. The temporal and spatial relationships of venous, arterial, and biliary lesions were studied to clarify their potential contributions to graft fibrosis. The severity, prevalence, and morphology of intimal lesions of vessels were analyzed and compared with the fibrosis stage. Three groups were found; group 1 (n = 5) with no hepatic vein (HV) lesions, group 2 (n = 5) with HV lesions only, and group 3 with lesions of both HV and portal veins (PV). The earliest lesion to develop, in 71% of grafts, was concentric intimal thickening of small HV. This was significantly more severe and frequent in grafts from group 3. With increasing frequency and severity of small HV sclerosis, fibrosis developed in medium/large veins. The morphology of larger vessel lesions suggested organized thrombus. Centrilobular fibrosis was significantly more severe in group 3 and developed unpredictably and sometimes rapidly. Conversely, portal fibrosis scores were significantly higher in grafts with ductular proliferation and did not correlate with venous lesions. This suggests that in CR, veno-occlusive-like lesions develop commonly in terminal hepatic venules, probably caused by immune-mediated damage. In only a proportion, with increased frequency and severity of the lesions, stasis and thrombosis in portal and larger veins occur and could result in loss of hepatic and portal venous outflow, which leads to ischemia and fibrosis. The stage of fibrosis did not correlate with foam-cell arteriopathy. A second pathway of portal fibrosis occurs in patients with longstanding biliary proliferation.

Clinicopathological analysis of liver allograft biopsies with late centrilobular necrosis: a comparative study in 54 patients.

BACKGROUND: Centrilobular necrosis (CLN) in liver allografts can be a difficult lesion to interpret histologically. Although long recognized in association with developing chronic rejection, recent studies have described the lesion in association with a number of other disease processes. To clarify the histologic features that could allow a specific diagnosis to be made and to determine the outcome in different diagnostic groups, we assessed biopsies from 54 patients with CLN. METHODS: Biopsies were classified as CLN with acute cellular rejection (ACR), CLN with hepatitis, CLN with developing chronic rejection (CR), and CLN of other etiology. Histologic features were assessed and then compared between groups, and clinical outcomes were noted. RESULTS: Discriminating features for the different groups were as follows: CLN and ACR showed bile duct injury, endothelialitis, and acinar congestion. CLN and CR showed severe bile duct injury, bile duct loss, or centrilobular swelling. CLN and hepatitis was often a diagnosis of exclusion, although interface hepatitis was more common in this group. Cases of autoimmune hepatitis usually demonstrated plasma cell predominance in the portal and acinar inflammatory infiltrate. Significantly, there was considerable overlap in the histologic features between the groups, accounting for the diagnostic difficulty. Patients in whom the CLN was associated with CR or vascular complications generally required retransplantation or died, but in the groups with ACR and hepatitis, the outcome was more favorable. CONCLUSIONS: With regard to most liver allograft biopsies showing late CLN, it is possible to make a specific diagnosis despite overlapping histologic features; this allows specific therapy to be instituted. Ultimately this is likely to contribute to improved graft survival.

Massive acinar cell apoptosis with secondary necrosis, origin of ducts in atrophic lobules and failure to regenerate in cyanohydroxybutene pancreatopathy in rats.

Cyanohydroxybutene (CHB), a glycosinolate breakdown product, causes pancreatic injury when given to animals in large amounts. To determine the course of CHB-induced pancreatopathy, rats were given a single subcutaneous dose of CHB and the pancreas weighed and examined by light and electron microscopy and immunohistochemistry at intervals from 2 h to 28 days. The pancreatic lesion was unusual in that there was marked early oedema with limited inflammatory cell infiltration, rapid synchronous onset of acinar cell apoptosis and early advanced atrophy engendering only a limited regenerative response. Acinar cell apoptosis was atypical in that cell fragmentation was limited and phagocytosis delayed, resulting in extensive secondary necrosis. As ducts were unaffected by CHB, the crowded ducts making up the epithelial component of atrophic lobules could be clearly shown to derive from their condensation and proliferation, not the redifferentiation of pre-existing acinar cells, widely held to produce this lesion. Although the basis of CHB selectivity and toxicity for pancreatic acinar cells remains unknown, the potential therapeutic benefit of such an agent in patients with pancreatitis or pancreatic tumours warrants further investigation.

Acinar cell apoptosis and the origin of tubular complexes in caerulein-induced pancreatitis.

The interrelationship between acinar cell apoptosis and tubular complex formation was examined in caerulein-induced pancreatitis using histology, immunohistochemistry, electron microscopy and DNA gel electrophoresis. Rats were given 8 hourly subcutaneous injections of caerulein, 24 micrograms/kg, for up to 2 days. Morphologically and biochemically typical apoptosis affected 4.6 and 8.9% of acinar cells at 1 and 2 days, respectively, resulting in removal of most acinar cells by 2 days. Consequently, pancreatic ducts, the lining cells expressing bcl-2 and therefore resistant to apoptosis, became much more closely approximated to form the basis of tubular complexes; small numbers of immunohistochemically discrete acinar cells in their lining were either pre-apoptotic resistant to it or newly formed. Proliferation of duct-like lining cells was associated with apoptosis, an increase in islet cells and acinar cell regeneration. There was evidence of duct to acinar cell differentiation but the main increase in acinar cell numbers appeared to derive from proliferation of newly formed acinar cells.

Elevated serum type IV collagen: a sensitive indicator of the presence of cirrhosis in haemochromatosis.

BACKGROUND/AIM: Hereditary haemochromatosis can now be diagnosed by genetic testing, although determining the presence or absence of cirrhosis remains crucial to patient management. While many studies have investigated the utility of various serum markers of cirrhosis in chronic liver diseases, few have examined specifically patients with hereditary haemochromatosis. The aim of this study was to assess the utility of serum type IV collagen and serum laminin in diagnosing hepatic fibrosis and cirrhosis in patients with hereditary haemochromatosis. METHODS: The study group consisted of 42 patients with hereditary haemochromatosis and 19 Caucasian controls. Serum type IV collagen, laminin, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase (TIMP-1) concentrations were measured by enzyme-linked immunosorbant assay in serum from patients with haemochromatosis and control subjects. Liver biopsies from patients with haemochromatosis were graded for fibrosis and correlated with serum markers of hepatic fibrosis. RESULTS: Serum type IV collagen concentration was significantly increased in haemochromatosis patients compared to controls (130+/-79 ng/ml vs 81 +/- 17 ng/ml, p<0.05) and was significantly correlated with both the grade of histological fibrosis (r=0.67, p<0.0001) and serum MMP-2 levels (r=0.42, p<0.05). A serum type IV collagen concentration > 115 ng/ml (mean+2 SD of controls) was 100% sensitive and 69% specific in detecting severe (grade 3) fibrosis and cirrhosis. The sensitivity results of serum laminin and TIMP-1 were 11% and 56% respectively. CONCLUSIONS: Elevated serum type IV collagen is a sensitive indicator of the presence of severe fibrosis and cirrhosis in patients with haemochromatosis. Useful markers of hepatic fibrosis in other chronic liver diseases may not be applicable to haemochromatosis.

Copper toxicosis in an Australian child.

A 20 month old Caucasian child living in rural Australia presented with liver failure after exposure from birth to milk formula made from acidic bore water containing excess copper leached from the copper piping. The liver pathology was identical to that seen in Indian childhood cirrhosis and similar disease in non-Indian children now termed idiopathic copper toxicosis (ICT). The only other Australian case, reported more than 20 years previously, had identical presentation, pathology and circumstances of occurrence. The rarity of ICT in Australia, despite a significant population at risk, and the implications of these cases are discussed.

Evidence for altered hepatic matrix degradation in genetic haemochromatosis.

BACKGROUND: Altered matrix degradation contributes to fibrosis in some liver diseases but the role of matrix degradation in fibrogenesis associated with genetic haemochromatosis has not previously been addressed. AIMS: To measure serum concentrations of tissue inhibitor of metalloproteinase 1 (TIMP-1) and matrix metalloproteinases (MMP), MMP-1, MMP-2, and MMP-3 in patients with haemochromatosis and control subjects. PATIENTS: Forty patients with haemochromatosis and 19 healthy control subjects. Ten of the 40 patients were studied before and after venesection therapy. METHODS: Serum levels of TIMP-1, MMP-1, MMP-2, and MMP-3 were measured by enzyme immunoassay and correlated to hepatic iron concentration and degree of histological fibrosis. RESULTS: Serum TIMP-1 was increased in patients with haemochromatosis compared with controls (163 (30) versus 123 (28) ng/ml, p < 0.0002). Mean serum TIMP-1 concentration of patients with haemochromatosis without fibrosis was significantly higher than in controls (153 (16) versus 123 (28) ng/ml, p = 0.03). Serum TIMP-1 concentration correlated with both hepatic iron concentration and hepatic iron index (r = 0.42, p < 0.01; r = 0.42, p < 0.01). Serum MMP-2 concentrations correlated with increasing degree of fibrosis in patients with haemochromatosis (r = 0.38, p = 0.01). The mean MMP-1: TIMP-1, MMP-2:TIMP-1 and age/sex matched MMP-3:TIMP-1 ratios were significantly lower in patients with haemochromatosis than controls (0.11 (0.06) versus 0.2 (0.14), p = 0.02; 3.32 (0.9) versus 3.91 (0.81), p = 0.05; and 0.26 (0.12) versus 0.47 (0.27), p = 0.007, respectively). Following venesection, MMP-2 and MMP-3 concentrations increased by 11% (p = 0.03) and 19% (p = 0.03), respectively. CONCLUSIONS: This study provides the first evidence of an alteration in matrix degradation in haemochromatosis that may be a contributing factor to hepatic fibrogenesis in this disease.

Functional and structural characterization of isolated perfused stingray liver including effects of ischaemia/reperfusion.

The morphological and functional characteristics of stingray liver were studied, including the effect of ischaemia/reperfusion. With an isolated perfused model, it was shown that the stingray liver was more resistant than the rat liver to ischaemia/reperfusion injury; this was consistent with the differing partial oxygen tensions usually present in the two species. This study confirmed that whereas stingray hepatocytes form tubules with central bile canaliculi as in other fish, the stingray liver has portal triads and a lobular architecture as in mammals. Apoptosis of hepatocytes, demonstrated in the normal liver, was only marginally enhanced by ischaemia/reperfusion. Resulting apoptotic bodies were phagocytized by macrophage-like cells in hepatocyte tubules. In contrast to rat liver, the stingray liver showed no necrosis after ischaemia-reperfusion.

Biochemical and molecular mechanisms regulating apoptosis.

In eukaryotes, the regulation of tissue cell numbers is a critical homeostatic objective that is achieved through tight control of apoptosis, mitosis and differentiation. While much is known about the genetic regulation of cell growth and differentiation, the molecular basis of apoptosis is less well understood. Genes involved in both cell proliferation and apoptosis reflect the role of some stimuli in both of these processes, the cell response depending on the overall cellular milieu. Recent research has given fascinating insights into the complex genetic and molecular mechanisms regulating apoptosis. A picture is emerging of the initiation in certain cells, after an apoptotic trigger, of sequential gene expression and specific signal transduction cascades that guide cells along the cell death pathway. Changes in gene expression precede the better known biochemical and morphological changes of apoptosis. It seems possible that, as a result of increased understanding of the cellular events preceding cell death, apoptosis may become more amenable to manipulation by appropriate drug- and gene-based therapies.

Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that occasionally progresses to cirrhosis but usually has a benign course. The aim of this study was to investigate the role of the hemochromatosis mutation Cys282Tyr in development of the mild hepatic iron overload found in some patients with NASH and its association with hepatic damage in these patients. METHODS: Fifty-one patients with NASH were studied. The presence of the Cys282Tyr mutation was tested in all patients, and the data were analyzed with respect to the histological grade of steatosis, inflammation, Perls' staining, hepatic iron concentration (HIC), and serum iron indices. RESULTS: Thirty-one percent of patients with NASH were either homozygous or heterozygous for the Cys282Tyr mutation. This mutation was significantly associated with Perls' stain grade (P < 0.005), HIC (P < 0.005), and transferrin saturation percentage (P < 0.005) but not with serum ferritin levels. Linear regression analysis showed that increased hepatic iron (Perls' stain or HIC) had the greatest association with the severity of fibrosis (P < 0.0001). CONCLUSIONS: The Cys282Tyr mutation is responsible for most of the mild iron overload found in NASH and thus has a significant association with hepatic damage in these patients. Heterozygosity for the hemochromatosis gene mutation therefore cannot always be considered benign.

Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction.

Acinar cell regeneration in the rat parotid gland after atrophy induced by a one week period of duct obstruction was examined using histology, immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemistry, antibodies to 5-bromo-2'-deoxyuridine (BrdU), injected one hour before tissue collection, and cytokeratin were employed. When clips were removed from the duct, only ductal epithelial cells remained; all acinar cells had been deleted. Some duct cells were BrdU positive. After three days, newly-formed acini comprising immature acinar cells had appeared; many of the cells were BrdU positive and mitotic figures were readily identified. Thereafter progressive acinar cell maturation and proliferation occurred, parotid gland weight returning to control levels by 7 days. Peak BrdU labelling indices for duct and acinar cells were on days 0 and 4, respectively. By TEM, cytoplasmic organelles in epithelial cells of transitional duct-acinar structures seen at 2 days were poorly developed. Immature acinar cells seen on day 3 contained zymogen granules and had increased endoplasmic reticulum and mitochondria. By day 5, maturing acinar cells had abundant endoplasmic reticulum and zymogen granules, resembling acinar cells in control glands. These observations indicated origin of acinar cell precursors from duct cells during regeneration of the acinar cell-free atrophic gland. Subsequent expansion of the acinar cell population was dependent on maturation and proliferation of these newly-formed cells.

Tumor necrosis factor-alpha induces apoptosis of human adipose cells.

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.

Hepatic stellate cell activation in genetic haemochromatosis. Lobular distribution, effect of increasing hepatic iron and response to phlebotomy.

BACKGROUND/AIMS: Activated hepatic stellate cells produce increased levels of collagen in animal models of chronic iron overload; however, their role in human genetic haemochromatosis is unknown. This study examined the relationship between hepatic iron concentration and hepatic stellate cell activation in genetic haemochromatosis. METHODS: Liver biopsies from 75 patients (55 with haemochromatosis, 14 haemochromatosis patients both pre- and post-phlebotomy and six non iron-loaded disease control subjects) were stained for iron using Perls' Prussian Blue. Thirty biopsies in which there was no evidence of either steatosis or inflammation were subjected to immunohistochemistry for alpha-smooth muscle actin and desmin and counterstained for iron. Forty-five biopsies demonstrated either steatosis or inflammation, in addition to excess iron. RESULTS: Stellate cells were identified by light microscopy as perisinusoidal cells containing numerous intracellular fat droplets. alpha-Smooth muscle actin was detected in biopsies with an hepatic iron concentration >60 micromol/g dry weight. Increasing hepatic iron concentration and hepatic iron index correlated with an increase in alpha-smooth muscle actin expression (r=0.81 and 0.72, respectively). Phlebotomy resulted in a significant decrease in alpha-smooth muscle actin expression. In early disease prior to histological evidence of collagen deposition, whilst activated stellate cells were located in Zone 1, greater numbers were found in Zones 2 and 3 distal to the region of heaviest iron overload. CONCLUSIONS: This study has demonstrated for the first time in humans a correlation between hepatic iron concentration and stellate cell activation in haemochromatosis, which is reversed by iron removal. Humoral factors from either iron-loaded hepatocytes or activated Kupffer cells may be responsible for early stellate cell activation in areas of the liver remote from heavy iron loading.

Large intestinal lymphocytic phlebitis and venulitis in chronic ulcerative colitis.

Colonic vasculitis is seen in Crohn's disease and as a component of primary systemic vasculitis. It has rarely been described in chronic ulcerative colitis. Here we report a case of ulcerative colitis with prominent transmural lymphocytic phlebitis and venulitis. Although this is, to our knowledge, the first description of such an association, its recognition is important if confusion with other entities is to be avoided. The etiology of the vascular changes is unclear but they may be a secondary phenomenon induced by antigens, toxins or cytokines draining from the inflamed mucosa.

An optimized model for rat liver perfusion studies.

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7. 2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are flow-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a flow rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g.

Chronic copper toxicosis presenting as liver failure in an Australian child.

A 20 month old Caucasian male child, after a five week illness, developed liver failure which was successfully treated by liver transplantation. The explanted liver had a histology identical to that seen in Indian childhood cirrhosis and its copper content was increased tenfold. Water used to prepare the child's milk feeds came from a bore via copper conduits and at times contained 120 mumol/l of copper, eight times the recommended maximum for human consumption. Because non-Indian cases of Indian childhood cirrhosis associated with excess copper ingestion are increasingly being recognised, and as early treatment can restore normal liver morphology, we support the use of the previously suggested alternative term for this condition, ie; 'copper-associated liver disease in childhood'. Measurement of hepatic copper concentrations in all children less than six years of age who develop hepatic failure of unknown cause will increase its recognition. On diagnosis sources of increased dietary copper should be investigated to ensure that younger siblings are not similarly exposed.

Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1.

The ability of beta cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic beta cell line, INS-1. Apoptosis was identified by detection of initial endonuclease-mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on beta cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of beta cell death is predominantly necrosis.