CD, or not CD, that is the question: a digital interobserver agreement study in coeliac disease.

OBJECTIVE: Coeliac disease (CD) diagnosis generally depends on histological examination of duodenal biopsies. We present the first study analysing the concordance in examination of duodenal biopsies using digitised whole-slide images (WSIs). We further investigate whether the inclusion of immunoglobulin A tissue transglutaminase (IgA tTG) and haemoglobin (Hb) data improves the interobserver agreement of diagnosis. DESIGN: We undertook a large study of the concordance in histological examination of duodenal biopsies using digitised WSIs in an entirely virtual reporting setting. Our study was organised in two phases: in phase 1, 13 pathologists independently classified 100 duodenal biopsies (40 normal; 40 CD; 20 indeterminate enteropathy) in the absence of any clinical or laboratory data. In phase 2, the same pathologists examined the (re-anonymised) WSIs with the inclusion of IgA tTG and Hb data. RESULTS: We found the mean probability of two observers agreeing in the absence of additional data to be 0.73 (±0.08) with a corresponding Cohen's kappa of 0.59 (±0.11). We further showed that the inclusion of additional data increased the concordance to 0.80 (±0.06) with a Cohen's kappa coefficient of 0.67 (±0.09). CONCLUSION: We showed that the addition of serological data significantly improves the quality of CD diagnosis. However, the limited interobserver agreement in CD diagnosis using digitised WSIs, even after the inclusion of IgA tTG and Hb data, indicates the importance of interpreting duodenal biopsy in the appropriate clinical context. It further highlights the unmet need for an objective means of reproducible duodenal biopsy diagnosis, such as the automated analysis of WSIs using artificial intelligence.

Portal pressure is of significant prognostic value in primary biliary cholangitis.

BACKGROUND AND AIMS: In other forms of chronic liver disease, measurement of portal pressure is of prognostic value, but this has not yet been established in primary biliary cholangitis (PBC). The aim of the study is to determine the prognostic value of hepatic venous pressure gradient (HVPG) in relation to liver-related survival outcomes, as well as to the development of hepatic decompensation, oesophageal varices and variceal bleeding. METHODS: Baseline HVPG and liver biopsies were obtained in 86 patients followed for 10 years in a controlled trial of colchicine treatment, and subsequently in a long-term observational cohort study for a further 30 years. RESULTS: There were 49 Hepatic deaths in addition to 10 Liver Transplants (Hepatic death/transplant; n = 59). Some of these were associated with a significant variceal bleed within 3 months of death or transplant (Portal hypertension-associated death or transplant; n = 19). There were 63 deaths from all causes. During follow-up, oesophageal varices developed in 26 patients, whilst 17 bled from varices and 32 developed hepatic decompensation over a median follow-up of 18.1 years (1.9-28.5). Baseline HVPG was highly predictive of all 6 clinical outcomes and contributed significant predictive information additional to that provided by Mayo score and Ludwig stage. CONCLUSION: Measurement of baseline portal pressure is of significant prognostic value in primary biliary cholangitis.

Advances in genetic technologies result in improved diagnosis of mismatch repair deficiency in colorectal and endometrial cancers.

BACKGROUND: Testing cancers for mismatch repair deficiency (dMMR) by immunohistochemistry (IHC) is a quick and inexpensive means of triaging individuals for germline Lynch syndrome testing. The aim of this study was to evaluate tumour dMMR and the prevalence of Lynch syndrome in patients referred to the Manchester Centre for Genomic Medicine, which serves a population of 5.6 million. METHODS: Tumour testing used IHC for MMR proteins with targeted BRAF and MLH1 promotor methylation testing followed by germline mutation and somatic testing as appropriate. RESULTS: In total, 3694 index tumours were tested by IHC (2204 colorectal cancers (CRCs), 739 endometrial cancers (ECs) and 761 other), of which 672/3694 (18.2%) had protein loss, including 348 (9.4%) with MLH1 loss. MLH1 loss was significantly higher for 739 ECs (15%) vs 2204 CRCs (10%) (p=0.0003) and was explained entirely by higher rates of somatic MLH1 promoter hypermethylation (87% vs 41%, p<0.0001). Overall, 65/134 (48.5%) patients with MLH1 loss and no MLH1 hypermethylation or BRAF c.1799T>A had constitutional MLH1 pathogenic variants. Of 456 patients with tumours showing loss of MSH2/MSH6, 216 (47.3%) had germline pathogenic variants in either gene. Isolated PMS2 loss was most suggestive of a germline MMR variant in 19/26 (73%). Of those with no germline pathogenic variant, somatic testing identified likely causal variants in 34/48 (71%) with MLH1 loss and in MSH2/MSH6 in 40/47 (85%) with MSH2/MSH6 loss. CONCLUSIONS: Reflex testing of EC/CRC leads to uncertain diagnoses in many individuals with dMMR following IHC but without germline pathogenic variants or MLH1 hypermethylation. Tumour mutation testing is effective at decreasing this by identifying somatic dMMR in >75% of cases.

Lynch syndrome for the gynaecologist.

KEY CONTENT: Lynch syndrome is an autosomal dominant condition closely associated with colorectal, endometrial and ovarian cancer.Women with Lynch syndrome are at increased risk of both endometrial and ovarian cancer and should be offered personalised counselling regarding family planning, red flag symptoms and risk-reducing strategies.Surveillance for gynaecological cancer in women with Lynch syndrome remains controversial; more robust data are needed to determine its effectiveness.Universal testing for Lynch syndrome in endometrial cancer is being adopted by centres across Europe and is now recommended by the National Institute for Health and Care Excellence; thus, gynaecologists must become familiar with testing strategies and their results.Testing strategies involve risk stratification of cancers based on phenotypical features and definitive germline testing. LEARNING OBJECTIVES: To define the pathogenesis of Lynch syndrome and its associated gynaecological cancers.To understand the testing strategies for Lynch syndrome in women with gynaecological cancer.To learn how best to counsel women with Lynch syndrome regarding gynaecological cancer and risk-reducing strategies to enable informed decision-making. ETHICAL ISSUES: Offering gynaecological surveillance despite a lack of robust evidence for its clinical effectiveness may falsely reassure women and delay risk-reducing hysterectomy.Genetic testing may yield variants of unknown significance with ill-defined clinical implications, which can lead to confusion and anxiety.Genetic testing has implications not only for the individual, but also for the whole family, so expert counselling is crucial.

Portal hypertension in primary biliary cholangitis: prevalence, natural history and histological correlates.

OBJECTIVES: The histopathological mechanisms underlying portal hypertension in primary biliary cholangitis (PBC) are poorly understood, as is its natural history. We have therefore determined the prevalence, severity and progression of portal hypertension in PBC and investigated whether its presence is related to specific histological lesions. METHODS: Hepatic venous pressure gradient (HVPG) was measured in 86 patients, with 186 assessments over up to 7 years of follow-up and the results correlated with a semiquantitative grading of 8 histological features and nodular regenerative hyperplasia (NRH). RESULTS: Portal hypertension (HVPG >5 mmHg) was present in 88% of all assessments (86% at baseline), and in 45% of patients at baseline was >12 mmHg (high-risk portal hypertension). The rise in portal pressure occurs early in the disease, since 45% of patients with normal serum bilirubin had a raised HVPG, as did 72% of patients with early (Ludwig stages 1 and 2) disease. After baseline, there was a small increase in HVPG over the next 5 years in most patients. In patients with precirrhotic PBC, 82% had portal hypertension and in 34% this was >12 mmHg. Portal pressure correlated significantly with a semiquantitative grading of cholestasis, interface hepatitis and portal tract and sinusoidal fibrosis. NRH was present in only 20% of wedge biopsies. CONCLUSIONS: Portal hypertension commences in the early stages of PBC, long preceding both rises in serum bilirubin and the development of cirrhosis. Around 34% of precirrhotic PBC patients have 'high-risk' portal hypertension, which is associated with lesions in the portal tracts and sinusoids rather than with NRH.

Assessment of mismatch repair deficiency in ovarian cancer.

BACKGROUND: Hereditary causes of ovarian cancer include Lynch syndrome, which is due to inherited pathogenic variants affecting one of the four mismatch repair genes involved in DNA repair. The aim of this study was to evaluate tumour mismatch repair deficiency and prevalence of Lynch syndrome in high-risk women referred to the Manchester Centre for Genomic Medicine with ovarian cancer over the past 20 years. METHODS: Women with ovarian cancer diagnosed before the age of 35 years and/or with a suggestive personal or family history of Lynch syndrome cancers underwent tumour testing with immunohistochemistry for mismatch repair deficiency and, where indicated, MLH1 promoter methylation testing followed by constitutional testing for Lynch syndrome. RESULTS: In total, 261 ovarian cancers were tested and 27 (10.3%; 95% CI 6.9% to 14.7%) showed mismatch repair deficiency by immunohistochemistry. Three of 7 with MLH1 loss showed MLH1 promoter hypermethylation, and 18 of the remaining 24 underwent constitutional testing for Lynch syndrome. A further 15 women with mismatch repair proficient tumours underwent constitutional testing because of a strong family history of Lynch syndrome cancers. Pathogenic variants were identified in 9/33 (27%) women who underwent constitutional testing, aged 33-59 years (median 48 years), including one whose tumour was mismatch repair proficient. Most Lynch syndrome tumours were of endometrioid histological subtype. CONCLUSIONS: Tumour mismatch repair deficiency identified by immunohistochemistry is a useful prescreen for constitutional testing in women with ovarian cancer with personal or family histories suggestive of Lynch syndrome.

The proportion of endometrial tumours associated with Lynch syndrome (PETALS): A prospective cross-sectional study.

BACKGROUND: Lynch syndrome (LS) predisposes to endometrial cancer (EC), colorectal cancer, and other cancers through inherited pathogenic variants affecting mismatch-repair (MMR) genes. Diagnosing LS in women with EC can reduce subsequent cancer mortality through colonoscopic surveillance and aspirin chemoprevention; it also enables cascade testing of relatives. A growing consensus supports LS screening in EC; however, the expected proportion of test positives, and optimal testing strategy is uncertain. Previous studies from insurance-based healthcare systems were limited by narrow selection criteria, failure to apply reference standard tests consistently, and poor conversion to definitive testing. The aim of this study was to establish the prevalence of LS and the diagnostic accuracy of LS testing strategies in an unselected EC population. METHODS AND FINDINGS: This was a prospective cross-sectional study carried out at a large United Kingdom gynaecological cancer centre between October 2015 and January 2017. Women diagnosed with EC or atypical hyperplasia (AH) were offered LS testing. Tumours underwent MMR immunohistochemistry (IHC), microsatellite instability (MSI), and targeted MLH1-methylation testing. Women <50 years, with strong family histories and/or indicative tumour molecular features, underwent MMR germline sequencing. Somatic MMR sequencing was performed when indicative molecular features were unexplained by LS or MLH1-hypermethylation. The main outcome measures were the prevalence of LS in an unselected EC population and the diagnostic accuracy of clinical and tumour testing strategies for risk stratifying women with EC for MMR germline sequencing. In total, 500 women participated in the study; only 2 (<1%) declined. Germline sequencing was indicated and conducted for 136 and 135 women, respectively. A total of 16/500 women (3.2%, 95% CI 1.8% to 5.1%) had LS, and 11 more (2.2%) had MMR variants of uncertain significance. Restricting testing to age <50 years, indicative family history (revised Bethesda guidelines or Amsterdam II criteria) or endometrioid histology alone would have missed 9/16 (56%), 8/13 (62%) or 9/13 (69%), and 5/16 (31%) cases of LS, respectively. In total 132/500 tumours were MMR deficient by IHC of which 83/132 (63%) had MLH1-hypermethylation, and 16/49 (33%) of the remaining patients had LS (16/132 with MMR deficiency, 12%). MMR-IHC with targeted MLH1-methylation testing was more discriminatory for LS than MSI with targeted methylation testing, with 100% versus 56.3% (16/16 versus 9/16) sensitivity (p = 0.016) and equal 97.5% (468/484) specificity; 64% MSI-H and 73% MMR deficient tumours unexplained by LS or MLH1-hypermethylation had somatic MMR mutations. The main limitation of the study was failure to conduct MMR germline sequencing for the whole study population, which means that the sensitivity and specificity of tumour triage strategies for LS detection may be overestimated, although the risk of LS in women with no clinical or tumour predictors is expected to be extremely low. CONCLUSIONS: In this study, we observed that age, family history, and histology are imprecise clinical correlates of LS-EC. IHC outperformed MSI for tumour triage and reliably identified both germline and somatic MMR mutations. The 3.2% proportion of LS-EC is similar to colorectal cancer, supporting unselected screening of EC for LS.

Liver biopsy in primary biliary cholangitis: is sinusoidal fibrosis the missing key?

AIMS: The role of liver biopsy in primary biliary cholangitis (PBC) is controversial, as is the optimal method of histological assessment. We compared the Ludwig and Ishak systems and three components of the Japanese (Nakanuma) staging system to evaluate their clinical and biochemical correlations and prognostic value. METHODS: We reviewed biopsies from 106 patients with PBC, derived from a previous trial of colchicine therapy with 24-34 years' follow-up, following which five clinical outcomes were evaluated: hepatic decompensation, cholestatic PBC death/liver transplant, portal hypertensive PBC death, all PBC deaths and overall survival. RESULTS: Ludwig and Ishak stages correlated well with prognostically significant parameters, including serum bilirubin, and both Mayo and Child Scores. Serum aspartate aminotransferase correlated with interface hepatitis (IFH), and alkaline phosphatase with orcein deposition, bile duct (BD) loss and cholestasis. Ludwig correlated with all five clinical outcomes, while Ishak stage was only significantly correlated with two. While sinusoidal fibrosis, orcein deposition, BD loss and cholestasis all predicted hepatic death/transplant, after correction for Mayo Score, the only histological parameters predictive of clinical outcomes were IFH (associated with two) and sinusoidal fibrosis (associated with all five). CONCLUSION: Liver biopsy is required in the diagnosis of around 20% of patients with PBC. The Ludwig system is of more prognostic value than both Ishak and any of the three individual components of the Nakanuma staging system, but the major histological parameter providing independent prognostic value beyond the Mayo Score is sinusoidal fibrosis.

Lynch syndrome screening in gynaecological cancers: results of an international survey with recommendations for uniform reporting terminology for mismatch repair immunohistochemistry results.

AIMS: Lynch syndrome (LS) is associated with an increased risk of developing endometrial carcinoma (EC) and ovarian carcinoma (OC). There is considerable variability in current practices and opinions related to screening of newly diagnosed patients with EC/OC for LS. An online survey was undertaken to explore the extent of these differences. METHODS AND RESULTS: An online questionnaire was developed by a panel of experts and sent to all members of the British Association of Gynaecological Pathologists (BAGP) and the International Society of Gynecological Pathologists (ISGyP). Anonymised results were received and analysed. Thirty-six BAGP and 44 ISGyP members completed the survey. More than 90% of respondents were aware of the association of LS with both EC and OC, but 34% were not aware of specific guidelines for LS screening. Seventy-one per cent of respondents agreed that universal screening for LS should be carried out in all newly diagnosed EC cases, with immunohistochemistry (IHC) alone as the preferred approach. Only 36% of respondents currently performed IHC or microsatellite instability testing on all newly diagnosed EC cases, with most of the remaining respondents practising selective screening, based on clinical or pathological features or both. A significant minority of respondents (35%) believed that patient consent was required before performance of mismatch repair (MMR) protein IHC. Almost all respondents favoured the use of standardised terminology for reporting MMR protein staining results, and this is proposed herein. CONCLUSION: There is wide support for universal LS screening in patients with EC, but this survey highlights areas of considerable variation in practice.

The Manchester International Consensus Group recommendations for the management of gynecological cancers in Lynch syndrome.

PURPOSE: There are no internationally agreed upon clinical guidelines as to which women with gynecological cancer would benefit from Lynch syndrome screening or how best to manage the risk of gynecological cancer in women with Lynch syndrome. The Manchester International Consensus Group was convened in April 2017 to address this unmet need. The aim of the Group was to develop clear and comprehensive clinical guidance regarding the management of the gynecological sequelae of Lynch syndrome based on existing evidence and expert opinion from medical professionals and patients. METHODS: Stakeholders from Europe and North America worked together over a two-day workshop to achieve consensus on best practice. RESULTS: Guidance was developed in four key areas: (1) whether women with gynecological cancer should be screened for Lynch syndrome and (2) how this should be done, (3) whether there was a role for gynecological surveillance in women at risk of Lynch syndrome, and (4) what preventive measures should be recommended for women with Lynch syndrome to reduce their risk of gynecological cancer. CONCLUSION: This document provides comprehensive clinical guidance that can be referenced by both patients and clinicians so that women with Lynch syndrome can expect and receive appropriate standards of care.

A prospective study of anal cancer screening in HIV-positive and negative MSM.

OBJECTIVE: The study sought to establish the feasibility and acceptability of anal screening among men MSM. DESIGN: Prospective cohort study. SETTING: Sexual health clinics in tertiary care. PATIENTS: Known HIV-positive and negative MSM who have anoreceptive intercourse. INTERVENTION: Anal screening with human papilloma virus (HPV) testing, liquid-based cytology and high-resolution anoscopy with biopsy of anoscopic abnormalities. Participants completed questionnaires at baseline and at 6 months. RESULTS: Anal HPV was highly prevalent in MSM (HIV-positive, 88% and HIV-negative, 78%). Despite the high prevalence of cytological abnormality in both HIV-positive (46.2%) and negative (35.0%) MSM, almost half of anal intraepithelial neoplasia (AIN) of all grades were associated with negative cytology. Anoscopically directed biopsies detected AIN3 or worse (AIN3+) in 14 of 203 (6.9%) of HIV-positive MSM and three of 81 (3.7%) HIV-negative MSM. The corresponding prevalence of AIN2+ was 26.6 and 20.9%, respectively. One case of AIN3 was detected at the second visit. Screening was considered to be highly acceptable by participants. CONCLUSION: The high prevalence of high-risk-HPV and frequency of false negative cytology in this study suggest that high-resolution anoscopy would have most clinical utility, as a primary screening tool for anal cancer in a high-risk group. The prevalence of AIN3+ in HIV-positive MSM lends support for a policy of screening this group, but the high prevalence of lower grade lesions which do not warrant immediate treatment and the limitations of treating high-grade lesions requires careful consideration in terms of a screening policy.

Impaired JNK signaling cooperates with KrasG12D expression to accelerate pancreatic ductal adenocarcinoma.

The c-Jun N-terminal protein kinase (JNK) and its two direct activators, namely the mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and MKK7, constitute a signaling node frequently mutated in human pancreatic ductal adenocarcinoma (PDAC). Here we demonstrate the cooperative interaction of endogenous expression of Kras(G12D) with loss-of-function mutations in mkk4 or both, mkk4 and mkk7 genes in the pancreas. More specifically, impaired JNK signaling in a subpopulation of Pdx1-expressing cells dramatically accelerated the appearance of Kras(G12D)-induced acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasias, which rapidly progressed to invasive PDAC within 10 weeks of age. Furthermore, inactivation of mkk4/mkk7 compromised acinar regeneration following acute inflammatory stress by locking damaged exocrine cells in a permanently de-differentiated state. Therefore, we propose that JNK signaling exerts its tumor suppressive function in the pancreas by antagonizing the metaplastic conversion of acinar cells toward a ductal fate capable of responding to oncogenic stimulation.

Administration of human recombinant activated protein C is not associated with pancreatic parenchymal haemorrhage in L-arginine-induced experimental acute pancreatitis.

CONTEXT: Microvascular thrombosis is a critical event in severe acute pancreatitis. Human recombinant activated protein C (Xigris®, Eli Lilly, Indianapolis, IN, USA) modulates the interplay between pro-inflammatory and pro-coagulant pathways and maintains microvascular patency. However, the anticoagulant properties of Xigris® may precipitate bleeding from the inflamed pancreas. OBJECTIVE: This study tests the hypothesis that Xigris® can ameliorate experimental acute pancreatitis without causing pancreatic haemorrhage. METHODS: Sprague Dawley rats were allocated as follows: Group 1: control (n=7); Group 2: acute pancreatitis (n=6); Group 3: administration of Xigris® 500 µg/kg body weight before induction of acute pancreatitis (n=6); and Group 4: Administration of Xigris® 500 µg/kg body weight 30 minutes after induction of acute pancreatitis (n=6). Acute pancreatitis was induced by intraperitoneal administration of L-arginine 300 mg/100 g body weight. Animals were sacrificed at 48 hours and biochemical, haematological, and histological markers of pancreatic haemorrhage and inflammation assessed. RESULTS: Median lipase in animals with acute pancreatitis was 10 U/mL (range: 7-16 U/mL) compared to 5.5 (range: 3-8 U/mL) in controls (P=0.028). Lipase was also elevated in animals given Xigris® both before (12 U/mL, range: 8-22 U/mL; P=0.031 vs. control group) and after (46 U/mL, range: 9-71 U/mL; P=0.015 vs. control group) induction of acute pancreatitis). Haemoglobin levels were similar among all groups (P=0.323). There was no histological evidence of pancreatic haemorrhage in animals treated with Xigris®. Pre-treatment with Xigris® was associated with a significant reduction in pancreatic injury. This effect was absent when Xigris® was administered after induction of acute pancreatitis. CONCLUSION: Xigris® did not lead to pancreatic haemorrhage in experimental acute pancreatitis. Administration of Xigris® prior to induction of acute pancreatitis was associated with amelioration of injury. This effect was not seen with administration of Xigris® after induction of acute pancreatitis.

Strategies for identifying hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the term given to a predisposition syndrome caused by inherited mutations in one of at least five DNA mismatch repair (MMR) genes. Dominant mutations in these genes predispose individuals to a range of cancers in addition to the most frequent, colorectal cancer. Endometrial cancer is the most notable additional malignancy, followed by ovarian, gastric, upper urethelial, and biliary cancers, and gliomas. Recognition of HNPCC is important so that targeted screening can be effected that will reduce the incidence of the main cancers. While such clinical criteria as Amsterdam and modified Amsterdam are reasonably specific, they lack sensitivity. Thus, tumor-related features have been used to improve sensitivity for identifying patients who can be selected for the relatively expensive direct mutation analysis of the various genes. Microsatellite instability (MSI) and loss of antibody staining for the proteins have been widely vaunted but have their own drawbacks. No one approach has received universal acceptance, and therefore adoption of one of perhaps three strategies, including clinical- and laboratory-based approaches, is still appropriate until an easier, quicker, and cheaper approach can be developed.

Oxidant stress in type I autoimmune hepatitis: the link between necroinflammation and fibrogenesis?

Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology characterized by circulating autoantibodies, hyperglobulinaemia and interface hepatitis. The mechanisms of progression from initial autoimmune attack to fibrosis and cirrhosis are unclear but oxidant stress may be involved. Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood and urine in 35 controls and in 33 patients with type-1 AIH; histology was assessed in 18 patients. In AIH, markers of lipid peroxidation were significantly elevated (8-isoprostane in both plasma and urine P < 0.001; plasma malondialdehyde P = 0.017). Total antioxidant capacity in protein-free serum and total glutathione in both whole blood and plasma were significantly reduced (P = 0.007, P = 0.037, P < 0.001, respectively). The antioxidants selenium, vitamin A and vitamin E were significantly decreased (P = 0.007, P < 0.001, P = 0.025, respectively); vitamin C was unchanged. Urinary 8-isoprostane correlated positively with interface hepatitis and necroinflammatory score and with hepatic fibrogenesis (type III procollagen peptide). Interface hepatitis correlated negatively with vitamin A and whole blood total glutathione. Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of AIH and provides a probable mechanism linking hepatic necroinflammation to fibrogenesis and disease progression.

Iron handling and gene expression of the divalent metal transporter, DMT1, in the kidney of the anemic Belgrade (b) rat.

BACKGROUND: We have previously shown that the rat kidney reabsorbs metabolically significant amounts of iron and that it expresses the divalent metal transporter 1, DMT1. The Belgrade (b) rat carries a mutation in DMT1 gene, which causes hypochromic, microcytic anemia due to impaired intestinal iron absorption and transport of iron out of the transferrin cycle endosome. In the duodenum of b/b rats, expression of DMT1 mRNA and protein is increased, suggesting a feedback regulation by iron stores. The aim of this study was to investigate iron handling and DMT1 expression in the kidneys of Belgrade rats. METHODS: Animals were maintained for 3 weeks on a synthetic diet containing 185 mg/kg iron (FeSO4), after which functional and molecular parameters were analyzed in male heterozygous (+/b) and homozygous (b/b) rats (N = 4 to 6 for each group). RESULTS: Serum iron concentration was significantly higher in b/b compared to +/b rats while urinary iron excretion rates were unchanged in b/b compared to +/b rats. Northern analysis using a rat DMT1 probe showed comparable mRNA levels between +/b and b/b animals. Western analysis and immunofluorescence microscopy performed using a polyclonal antibody against rat DMT1 showed that DMT1-specific immunoreactivity was almost absent in the kidneys of b/b rats compared to that seen in +/b animals. CONCLUSION: Our results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.

Expression of nitric oxide synthase isoforms in human liver cirrhosis.

Several mediators of systemic vasodilatation in liver cirrhosis have been reported. Among these is nitric oxide (NO), which has been proposed as one of the main mediators. In this study, sera and liver biopsies were analysed from 15 patients with clinically and pathologically diagnosed liver cirrhosis. In addition, sera from seven and liver biopsies from three healthy controls were used. Serum levels of nitrite (the end product of NO) were measured using the Griess reaction and the expression of the inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (ecNOS) proteins was investigated using immunohistochemistry. This study shows that serum nitrite levels (94 +/- 9.8 micro mol/l) in cirrhotic patients were significantly (p < 0.05) increased in comparison with the controls (36.6 +/- 11.03 micro mol/l). iNOS was completely absent from the control group but was highly expressed in the livers from the cirrhotic group. iNOS was seen mainly in the inflammatory cells infiltrating the portal tracts, blood monocyte-like cells, hepatocytes, sinusoidal cells, and endothelial cells. However, expression of ecNOS was only seen in the vascular endothelial cells of both the control and the cirrhotic groups, but was much higher in the latter. It is therefore clear that NO is augmented in cirrhotic patients and it is mainly produced by induction of iNOS. Moreover, NO up-regulation is dependent on the inflammatory stage of liver cirrhosis. ecNOS production could be a normal chronic adaptation mechanism of the endothelium to the chronically increased splanchnic blood flow secondary to portal hypertension. In the near future, the appropriate inhibition of NO activity by using NOS-active agents may provide a novel strategy for the treatment of patients with liver cirrhosis.

Altered dietary iron intake is a strong modulator of renal DMT1 expression.

Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an iron-enriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate.

Oxidant stress is a significant feature of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disorder characterised by an immunological, and often granulomatous, attack on bile ducts leading to fibrosis, cirrhosis, liver failure and death. Animal and human studies suggest that oxidant stress plays a key role in progression of other liver diseases, but no comprehensive investigation has been performed previously in PBC. A wide range of lipid peroxidation and antioxidant markers were measured in the blood and urine of 41 patients with histologically confirmed PBC. Lipid peroxidation markers were significantly elevated [plasma and urinary 8-isoprostane, P<0.001; plasma malondialdehyde (MDA), P=0.007] compared to age- and sex-matched controls. The most striking antioxidant depletion occurred with plasma total glutathione where levels were significantly reduced (30% of controls). Total serum antioxidant levels were decreased (P=0.013) and serum selenium and vitamin A were also lower (both P<0.001); vitamins C and E were normal. Most patients had early disease biochemically and were Child-Pugh grade A. Urinary 8-isoprostane correlated positively with Ludwig stage and markers of hepatic injury and cholestasis. This study clearly demonstrates that oxidant stress, as reflected in a comprehensive spectrum of lipid peroxidation and antioxidant markers, is a significant feature of early-stage PBC.

Oxidative stress in chronic hepatitis C: not just a feature of late stage disease.

BACKGROUND/AIMS: Chronic hepatitis C infection is a major world-wide problem, frequently progressing to cirrhosis, liver failure or hepatoma. The pathological mechanisms of disease progression are unclear but oxidant stress may play a role. METHODS: Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood or urine from 42 chronic hepatitis C patients. Fibrosis was graded histologically in a subgroup of 33 patients. RESULTS: The lipid peroxidation marker 8-isoprostane and the ratio of oxidized to reduced glutathione were significantly elevated (P<0.001, P=0.006). The antioxidants glutathione, selenium and vitamins A, C and E were significantly decreased (all P<0.001) compared to age and sex matched controls. Abnormal values were more marked in cirrhotics, but significant changes were also observed in the non-cirrhotic group. The fibrosis score correlated positively with urinary 8-isoprostane and type III procollagen peptide and negatively with vitamin A. CONCLUSIONS: Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of hepatitis C infection. Although more severe in the cirrhotic group, there was clear evidence of oxidant stress in non-cirrhotic patients. Antioxidant therapy may therefore have a role in slowing disease progression to cirrhosis.