Neal I. Walker

Clinical and Endoscopic Features of Eosinophilic Esophagitis in Adults

BACKGROUND Eosinophilic esophagitis in adults is regarded as unusual, being diagnosed mostly in young men presenting with dysphagia. Mucosal furrows are a sentinel endoscopic feature. This study examined the demographic and clinical profile of adults with eosinophilic esophagitis seen from 1981 to 2002. METHODS All patients from an Australian provincial city (population 198,000) with otherwise unexplained eosinophilic inflammation of the squamous epithelium (>/=30 eosinophils per high-power field) were included in a retrospective review. RESULTS A diagnosis of eosinophilic esophagitis was made in 31 patients (24 men, 7 women; mean age 34 years, range 14-77 years). The diagnosis was made in 19 (61%) of the 31 patients during the most recent 2 years (none between 1981 and 1994 vs. 12 between 1995-2000 vs. 19 between 2000-2001). Esophageal mucosal furrows were present in 30 (97%), a finding infrequently recognized before 2001. Dysphagia was documented in 26 (89%). Symptoms had been present for long periods before diagnosis (mean 54 months; range 0-180 months), and diagnosis was delayed in 7 (mean 81 months, range 20-144 months) because sentinel features were overlooked at endoscopy. Strictures, often evident only as a result of mucosal shearing during dilation, were present in 17 (57%). Esophageal dilation preformed in 17 (mean 3.4 dilations per patient, range 1-13) consistently relieved symptoms; tears were recorded in 13 (87%), but no serious complication resulted. CONCLUSIONS Eosinophilic esophagitis in adults of all ages is more common than recognized. Mucosal furrows are easily overlooked at endoscopy although this finding is an important clue to diagnosis. Strictures, a frequent consequence, can be safely managed by dilation.

Tumor Necrosis Factor-α Induces Apoptosis of Human Adipose Cells

Tumor necrosis factor-α (TNF-α) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-α mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-α 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-α in limiting increase in fat mass. The aim of the present study was to determine if TNF-α 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-α 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-α induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-α induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-α 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-α.

Only patients with dysplasia progress to adenocarcinoma in Barrett's oesophagus.

Columnar lined oesophagus (Barretts oesophagus) carries a risk for the development of adenocarcinoma. Epithelial dysplasia appears to be a precursor but the utility of this marker for predicting subsequent adenocarcinoma is unsettled. We therefore prospectively studied 81 patients with histologically proven columnar epithelium of at least the distal 3 cm of the tubular oesophagus with regular endoscopic biopsies for a total of 289.2 patient years (mean 3.6 years, range 0.5-8). Twenty three patients (28%) had epithelial dysplasia detected during follow up. Both patients with persistent high grade dysplasia present on initial biopsies developed adenocarcinoma after 2.6-4.5 years, despite the absence of gross macroscopic change. The initial single layer pleomorphic high grade dysplasia in one patient regressed to low grade dysplasia which has persisted for 1.5 years. Of 10 patients with initial low grade dysplasia, one progressed to adenocarcinoma in 4.3 years. The low grade dysplasia persisted unchanged in seven patients for 1.5-7 years and appears to have regressed in two patients after three to five years. Ten patients developed low grade dysplasia during the surveillance period. This has persisted unchanged in six patients from 0.5-5 years, regressed in three for 0.5-5 years and has appeared after the first yearly biopsy in one patient. No patient without dysplasia has developed adenocarcinoma. The incidence of adenocarcinoma in Barretts oesophagus in this study is one case per 96 patient years. This is 61 times (95% confidence limits 12-176) the age adjusted incidence of oesophageal cancer in Australia. Persistent high grade dysplasia appears to be a sensitive indicator for the development of subsequent adenocarcinoma.

The serrated pathway to colorectal carcinoma: current concepts and challenges

Approximately 30% of colorectal carcinomas develop via a serrated neoplasia pathway, named for the pattern of crypts in the precursor polyps. Molecular abnormalities consistently involve methylation of CpG islands [CpG island methylator phenotype (CIMP)] of low degree (CIMP‐L) or high degree (CIMP‐H), and activating mutations of the mitogen‐activated protein kinase pathway components BRAF or KRAS. Microsatellite instability (MSI) of a high level (MSI‐H) is often present, allowing for a molecular classification of serrated pathway carcinoma as: (i) BRAF mutant/CIMP‐H with either a) MSI‐H or b) microsatellite stable (MSS); and (ii) KRAS mutant/CIMP‐L/MSS. Precursor polyps include sessile serrated adenoma (SSA), characterized by proximal location, crypt architectural disturbance, and BRAF mutation. Microvesicular hyperplasic polyp (MVHP) probably precedes the development of SSA, and borderline lesions between MVHP and SSA occur. Cytological dysplasia in SSA portends advanced genetic abnormality and a high risk of progression to carcinoma. The traditional serrated adenoma has a predilection for the left colon, tubulovillous architecture, eosinophilic cytoplasm, and frequent KRAS mutation. Serrated morphology carcinoma is a new World Health Organization subtype with well‐differentiated, mucinous or trabecular patterns. It has frequent KRAS or BRAF mutations and a poor prognosis. This review provides an insight into the histology and molecular mechanisms driving these serrated pathway lesions.

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

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.

Leptin and the risk of Barrett’s oesophagus

Background: Obesity is associated with increased risks of Barrett’s oesophagus and oesophageal adenocarcinoma. Alterations in serum leptin and adiponectin, obesity-related cytokines, have been linked with several cancers and have been postulated as potential mediators of obesity-related carcinogenesis; however, the relationship with Barrett’s oesophagus remains unexplored. Methods: Serum leptin and adiponectin concentrations were measured on two subsets of participants within a case–control study conducted in Brisbane, Australia. Cases were people aged 18–79 years with histologically confirmed Barrett’s oesophagus newly diagnosed between 2003 and 2006. Population controls, frequency matched by age and sex to cases, were randomly selected from the electoral roll. Phenotype and medical history data were collected through structured, self-completed questionnaires. Odds ratios (OR) and 95% CI were calculated using multivariable logistic regression analysis. Results: In the pilot analysis (51 cases, 67 controls) risks of Barrett’s oesophagus were highest among those in the highest quartile of serum leptin (OR 4.6, 95% CI 0.6 to 33.4). No association was seen with adiponectin. In the leptin validation study (306 cases, 309 controls), there was a significant threefold increased risk of Barrett’s oesophagus among men in the highest quartile of serum leptin (OR 3.3, 95% CI 1.7 to 6.6) and this persisted after further adjustment for symptoms of gastro-oesophageal reflux (OR 2.4, 95% CI 1.1 to 5.2). In contrast, the risk of Barrett’s oesophagus among women decreased with increasing serum leptin concentrations. Conclusions: High serum leptin is associated with an increased risk of Barrett’s oesophagus among men but not women. This association is not explained simply by higher body mass or gastro-oesophageal reflux among cases. The mechanism remains to be determined.

Predicting Cholangiocarcinoma in Patients with Primary Sclerosing Cholangitis Before Transplantation

Patients with primary sclerosing cholangitis are at an increased risk of developing cholangiocarcinoma, which is difficult to diagnose because the biliary tree is already distorted. Eleven patients with primary sclerosing cholangitis who underwent orthotopic liver transplantation at this hospital were evaluated. Four patients had coincidental histologically proved cholangiocarcinoma. Patients with cholangiocarcinoma in contrast to patients without tumour presented with rapid onset of persistent jaundice, pruritus, and weight loss associated with an appreciable rise in bilirubin (8x v 2x) and alkaline phosphatase (3.5x v 1.2x) over one year. Cholangiography and computed tomography showed appreciably dilated intrahepatic bile ducts (3/4 v 0/7). The diagnosis of cholangiocarcinoma could only be established before operation in one patient by fine needle aspiration cytology. Tumour was recognised at operation in one other. Histological examination of hepatectomy specimens showed that patients with cholangiocarcinoma had less advanced histological features of primary sclerosing cholangitis. Multiple areas of carcinoembryonic antigen positive epithelial atypia and carcinoma in situ were found in all patients with cholangiocarcinoma. Cholangiocarcinoma recurred in two patients at 14 and 39 months after transplantation. Superimposed cholangiocarcinoma can be predicted in most patients with cholangitis before transplantation, although a definitive diagnosis is difficult to make. Their prognosis after successful transplantation is guarded.

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

The ability of ß 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 ß 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 ß cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of ß cell death is predominantly necrosis.

Fatal Reactivation of Precore Mutant Hepatitis-B Virus-Associated with Fibrosing Cholestatic Hepatitis After Bone-Marrow Transplantation

Liver failure caused by reactivation of hepatitis B virus (HBV) is an uncommon complication of bone marrow transplantation [1-3]. Fibrosing cholestatic hepatitis is a recently described liver lesion that develops after liver transplantation for chronic HBV infection. Hepatitis B virus precore mutant infection has been associated with liver failure after cytotoxic chemotherapy [4] and with fibrosing cholestatic hepatitis after liver transplantation [5, 6], but the relation is unclear. We describe a patient who developed fibrosing cholestatic hepatitis and HBV precore mutant infection after bone marrow transplantation. Case Report A 49-year-old Chinese man had allogeneic bone marrow transplantation for acute myeloid leukemia. The patient had no history or clinical or biochemical evidence of liver disease. Before transplantation, the patient was positive for the hepatitis B surface antigen (HBsAg), negative for the hepatitis B e antigen (HBeAg), positive for the antibody to HBe (anti-HBe), positive for the antibody to hepatitis B core IgG (anti-HBc), and negative for the antibody to hepatitis B surface antigen (anti-HBs). Serology was unchanged throughout the course of therapy after transplantation. The human leukocyte antigen-identical donor was his healthy 43-year-old brother, whose only HBV marker was IgG anti-HBc. Hepatitis B virus could not be sequenced before transplantation because both donor and recipient were HBV-DNA-negative by polymerase chain reaction [7]. The patient received standard prophylaxis for graft-versus-host disease with methotrexate and cyclosporine. Hematologic remission was verified by bone marrow examinations on days 100 and 190. The hepatologic course is shown in Figure 1. Early complications included transient veno-occlusive disease and grade 2 graft-versus-host disease associated with modest elevation of liver test findings. Serum on day 55 was positive for HBV DNA by polymerase chain reaction. Figure 1. Hepatologic course after bone marrow transplantation, including alanine aminotransferase (ALT) levels and hepatitis B virus (HBV) DNA. Successful treatment with ganciclovir for cytomegalovirus gastritis and pericarditis was initiated after day 94. On day 143, prednisone was given for recurrent jaundice and neutropenia. Liver function progressively deteriorated, and serum HBV DNA increased. Examination of liver histologic findings on day 171 showed early fibrosing cholestatic hepatitis with severe swelling of hepatocytes, apoptotic bodies, intense hepatocyte immunohistochemical staining for hepatitis B core antigen (HBcAg) and HBsAg but little inflammation (Figure 2). On day 173, therapy with cyclosporine was stopped, and therapy with ganciclovir, 5 mg/kg daily, and foscarnet, 5.4 g daily, was initiated [5]. Liver failure progressed, and the patient died on day 198. Autopsy was refused. Hepatitis B virus DNA from serum was amplified by polymerase chain reaction for direct sequencing (Applied Biosystems, Scoresby, Victoria, Australia) of the precore region. The precore mutant HBV (guanosine to adenosine substitution at nucleotide 1896) was present throughout the course of therapy after transplantation. Figure 2. Immunohistochemical stains for hepatitis B surface antigens (top) and core antigens (bottom) on day 171. Discussion Severe reactivation of hepatitis B in HBsAg-positive persons after bone marrow transplantation is infrequent and unpredictable [1-3, 8, 9]. Of 1980 patients who had bone marrow transplantation surveyed in the published series, 38 were HBsAg-positive before transplantation. Hepatitis B e antigen was present in the three reported cases of fatal reactivation (two HBsAg-positive patients, one anti-HBs-positive and anti-HBc-positive patient) [1-3]. In our patient, reactivation was not detected by standard serologic testing; HBeAg did not reappear, and anti-HBc IgM was not present. This is the phenotype of precore mutant HBV. The mutation in the precore region creates a translational stop codon, which results in failure to secrete HBeAg. This serologic finding is described in a report of three cases of fulminant hepatic failure from precore mutant HBV after cytotoxic chemotherapy [4]. It is clear from the literature and from our case that HBV DNA must be measured to detect reactivation of precore mutant HBV. Earlier detection is reported to facilitate successful antiviral treatment after liver transplantation [5]. Donor serology is important for the outcome. In the published series, 8 of 38 HBsAg-positive patients lost HBsAg, and 3 patients seroconverted to anti-HBs [1-3, 8, 9]. In the latter cases, the donors were anti-HBs-positive. Adoptive transfer of immunity to HBV is well documented [10]. Vaccination of our patients donor (who was anti-HBc-positive) before bone marrow harvest may have evoked anti-HBs and resulted in the elimination of HBV by the graft. To our knowledge, this is the first report of fibrosing cholestatic hepatitis after bone marrow transplantation. In immunocompetent hosts, hepatitis B-induced liver disease is caused by an immune system attack on infected hepatocytes. Fibrosing cholestatic hepatitis, a distinct and usually fatal form of hepatitis B-associated liver injury, is described in patients receiving immunosuppressive therapy after liver transplantation. Hepatocytes accumulate large amounts of HBsAg and HBcAg, which appear to be cytopathic [6]. Because the precore mutant is unable to secrete HBeAg from the liver cell, we propose that it plays a role in the accumulation of HBV antigens in fibrosing cholestatic hepatitis. Our patient showed the unique combination of fatal reactivation of precore mutant HBV after bone marrow transplantation and fibrosing cholestatic hepatitis. This case highlights the need to detect HBV precore mutant reactivation after bone marrow transplantation and the need for earlier antiviral treatment.

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.