Quantifying the hygroscopic properties of cyclodextrin containing aerosol for drug delivery to the lungs.

Aerosol dynamics is important to quantify in drug delivery to the lungs with the aim of delivering therapeutics to a target location and optimising drug efficacy. The macrocycle (2-hydroxypropyl)-ß-cyclodextrin (2-HP-ß-CD) is thought to alleviate symptoms associated with neurodegenerative diseases when inhaled but the hygroscopic response is not well understood. Here we measure the hygroscopic growth of individual aqueous aerosol containing 2-HP-ß-CD in optical tweezers through analysis of morphology-dependent resonances arising in Raman spectra. Droplets are analysed in the size range of 3-5 µm in radius. The evolving radius and refractive index of each droplet are measured in response to change in relative humidity from 98-20% to determine mass and radius based hygroscopic growth factors, and compared with dynamic vapour sorption measurements. Bulk solution refractive index and density measurements were used in accordance with the self-consistent Lorenz-Lorentz rule to determine melt solute and droplet properties. The refractive index of 2-HP-ß-CD was determined to be 1.520 ± 0.002 with a density of 1.389 ± 0.005 g cm-3. To our knowledge, we show the first aerosol measurements of 2-HP-ß-CD and determine hygroscopicity. By quantifying the hygroscopic growth and physicochemical properties of 2-HP-ß-CD, the impact of aerosol dynamics can be accounted for in tailoring drug formulations and informing models used to predict drug deposition patterns within the respiratory system.

Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.

BACKGROUND & AIMS: Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD. METHODS: PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD. RESULTS: Genotype frequencies were significantly different between NAFLD-HCC cases (CC=28, CG=43, GG=29) and NAFLD-controls (CC=125, CG=117, GG=33) (p=0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23-4.14], p=0.0082), with GG homozygotes exhibiting a 5-fold [1.47-17.29], p=0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n=1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55-4.10], p=0.0002; GG vs. CC: OR 12.19 [6.89-21.58], p<0.0001). CONCLUSIONS: Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

A lipid to treat non-alcoholic fatty liver disease - the dawn of 'lipo-rehabilitation'?

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl sidechains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatictriglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Genetic variants regulating insulin receptor signalling are associated with the severity of liver damage in patients with non-alcoholic fatty liver disease.

BACKGROUND/AIMS: The aim of this study was to assess the effect of functional ENPP1(ectoenzyme nucleotide pyrophosphate phosphodiesterase 1)/PC-1 (plasma cell antigen-1) and IRS-1 (insulin receptor substrate-1) polymorphisms influencing insulin receptor activity on liver damage in non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, whose progression is associated with the severity of insulin resistance. PATIENTS AND METHODS: 702 patients with biopsy-proven NAFLD from Italy and the UK, and 310 healthy controls. The Lys121Gln ENPP1/PC-1 and the Gly972Arg IRS-1 polymorphisms were evaluated by restriction analysis. Fibrosis was evaluated according to Kleiner. Insulin signalling activity was evaluated by measuring phosphoAKT levels by western blotting in a subset of obese non-diabetic patients. RESULTS: The ENPP1 121Gln and IRS-1 972Arg polymorphisms were detected in 28.7% and 18.1% of patients and associated with increased body weight/dyslipidaemia and diabetes risk, respectively. The ENPP1 121Gln allele was significantly associated with increased prevalence of fibrosis stage >1 and >2, which was higher in subjects also positive for the 972Arg IRS-1 polymorphism. At multivariate analysis, the presence of the ENPP1 121Gln and IRS-1 972Arg polymorphisms was independently associated with fibrosis >1 (OR 1.55, 95% CI 1.24 to 1.97; and OR 1.57, 95% CI 1.12 to 2.23, respectively). Both polymorphisms were associated with a marked reduction of approximately 70% of AKT activation status, reflecting insulin resistance and disease severity, in obese patients with NAFLD. CONCLUSIONS: The ENPP1 121Gln and IRS-1 972Arg polymorphisms affecting insulin receptor activity predispose to liver damage and decrease hepatic insulin signalling in patients with NAFLD. Defective insulin signalling may play a causal role in the progression of liver damage in NAFLD.

Abnormal serum alanine aminotransferase levels are associated with impaired insulin sensitivity in young women with polycystic ovary syndrome.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) and polycystic ovary syndrome (PCOS) are both associated with insulin resistance.We assessed whether NAFLD is associated with impaired insulin sensitivity in PCOS women independently of age and total adiposity. SUBJECTS AND METHODS: We enrolled 14 young PCOS women with NAFLD, 14 women with PCOS alone and 14 healthy controls, who were matched for age, body mass index, and total body fat (by bio-impedance analyzer). NAFLD was diagnosed by the surrogate measure of abnormal serum alanine aminotransferase (ALT) concentrations (defined as ALT>19 U/l) after excluding other secondary causes of liver disease (alcohol, virus, and medications). Insulin sensitivity was measured by euglycemic hyperinsulinemic clamp. RESULTS: Insulin sensitivity was markedly decreased (p<0.001) in PCOS women with abnormal ALT levels, whereas it was similar between PCOS women with normal ALT levels andmatched healthy controls (8.3+/-2.5 vs 12.1+/-1.7 vs 13.2+/-1.8 mg/min x kg of fat-free mass, respectively). PCOS women with abnormal ALT levels also had higher plasma triglycerides and lower HDLcholesterol concentrations than those with PCOS alone. There was a strong inverse association between serum ALT levels and insulin sensitivity in the whole group of PCOS women (r=-0.59, p=0.0013). CONCLUSIONS: Abnormal serum ALT levels, as surrogate measure of NAFLD, are closely associated with impaired insulin sensitivity in young PCOS women in a manner that is independent from the contribution of age and total adiposity. Early recognition of NAFLD by radiological imaging tests in this group of young patients is warranted.

Endovascular repair of the thoracic aorta: predictors of 30-day mortality in patients on the New Zealand Thoracic Aortic Stent Database (NZ TAS).

OBJECTIVES: To evaluate data in the New Zealand Thoracic Aortic Stent database to try and identify a scoring system that could predict 30-day mortality in patients undergoing stenting of the descending thoracic aorta (TEVAR). DESIGN: Retrospective analysis of the New Zealand thoracic aortic stent database between December 2001 and August 2007. MATERIALS AND METHODS: The 30-day mortality of the 122 patients is 7.38% (n=9). Risk factors were recorded based on the Society of Thoracic Surgeons (STS) risk score. Glasgow aneurysm score was calculated and the pathology being treated analysed. Univariate analysis was carried out. RESULTS: The mortality of three pathology groups was compared. 30-day mortality was 2.04% (n=1) in the elective aneurysm group, 17.95% (n=7) in the complicated Stanford type B dissection group, and 0% (n=0) in the trauma group. Thirty-day mortality is significantly higher in the dissection group compared with the elective aneurysm (p=0.02) and trauma (p=0.03) groups. The most frequent risk factors in the dissection group of patients were peripheral vascular disease, smoking and hypertension. Although percentage mortality is higher with increasing GAS, the results are not statistically significant (p=0.34). No independent risk factors were identified from the STS risk score data. CONCLUSION: No specific risk score system seems to be able to predict mortality in TEVAR patients.

Outcomes of endovascular repair of acute thoracic aortic injury: interrogation of the New Zealand thoracic aortic stent database (NZ TAS).

OBJECTIVES: Report the New Zealand national experience of endovascular repair of acute traumatic thoracic aortic injuries (TTAIs). DESIGN: Retrospective analysis of the New Zealand thoracic aortic stent database between December 2001 and December 2007. MATERIALS AND METHODS: Of the 134 patients on the database, 27 patients (20%) underwent endovascular repair of TTAI. Data collected included age, sex, cause of injury, details of the procedure, complications and mortality. RESULTS: Most patients were young, median age 20 (15-78), male (n=19, 70%), and involved in motor vehicle accidents (n=23, 85%). Median length of aorta stented was 117 mm (77-200 mm). Great vessel origins were covered intentionally in 23 (85%) patients, four (17%) requiring a hybrid procedure. Average procedure time was 98.3 min (35-180). Primary technical success was 96%, secondary technical success 100%. Endoleaks were observed in four (15%) patients, one requiring a second endovascular procedure. There were no conversions to open surgery. Procedure-related complications occurred in four (15%) patients. No patient developed cord injury. All cause mortality at 30 days is one (4%) and at discharge is two (7%) due to associated poly-trauma. CONCLUSION: Our results add further evidence of the safety of endovascular repair of thoracic aortic injury and compare favourably with those of other centres.

Fatigue in non-alcoholic fatty liver disease (NAFLD) is significant and associates with inactivity and excessive daytime sleepiness but not with liver disease severity or insulin resistance.

OBJECTIVE: To quantify fatigue in non-alcoholic fatty liver disease (NAFLD), to determine whether perceived fatigue reflects impairment of physical function and to explore potential causes. PATIENTS AND METHODS: A cohort study was carried out on 156 consecutive patients with histologically proven NAFLD studied in two cohorts. Phase 1 determined the perceived fatigue experienced by NAFLD patients (assessed using the Fatigue Impact Scale (FIS)) in comparison with normal and liver disease controls, and the relationship to physical function (actigraphy). In phase 2, biological associations of fatigue in NAFLD were explored. RESULTS: Fatigue was markedly higher in NAFLD patients than in controls (mean (SD) FIS 51 (38) vs 8 (12), p<0.001). NAFLD patients showed significantly lower physical activity over 6 days (7089 (2909) mean steps/day vs 8676 (2894), p = 0.02). A significant inverse correlation was seen between FIS and physical activity (r = 0.1, p = 0.02). Fatigue experienced by NAFLD patients was similar to that in primary biliary cirrhosis (n = 36) (FIS 64 (9) vs 61 (2), p = NS). No association was seen between FIS and biochemical and histological markers of liver disease severity or insulin resistance (homeostasis model assessment (HOMA)) (r < 0.005). Significant association was seen between fatigue severity and daytime somnolence (Epworth Sleepiness Scale) (r = 0.2, p < 0.001). CONCLUSION: Fatigue is a significant problem in NAFLD, is similar in degree to that in primary biliary cirrhosis patients and is associated with impairment in physical function. Fatigue in NAFLD appears to be unrelated to either severity of underlying liver disease or insulin resistance, but is associated with significant daytime somnolence.

The Glasgow alcoholic hepatitis score identifies patients who may benefit from corticosteroids.

INTRODUCTION: There is no consensus on the pharmacological treatment of alcoholic hepatitis. The Glasgow alcoholic hepatitis score (GAHS) has been shown to be more accurate than the modified Maddrey's discriminant function (mDF) in the prediction of outcome from alcoholic hepatitis. This study aimed to determine whether the GAHS was able to identify those patients who would benefit from corticosteroids. METHODS: 225 patients with an mDF greater than or equal to 32 from five hospital centres in the United Kingdom were reviewed. Patient survival relative to the GAHS and the use of corticosteroids was recorded. RESULTS: 144 patients with an mDF greater than or equal to 32 (64%) also had a GAHS greater than or equal to 9. There was no difference in survival between untreated or corticosteroid-treated patients for those with a GAHS less than 9. For patients with a GAHS greater than or equal to 9 the 28-day survival for untreated and corticosteroid-treated patients was 52% and 78% (p = 0.002), and 84-day survival was 38% and 59% (p = 0.02), respectively. CONCLUSIONS: Among patients with an mDF greater than or equal to 32, there was no appreciable benefit from treatment with corticosteroids in patients with a GAHS less than 9. Patients with a GAHS greater than or equal to 9 have an extremely poor prognosis if they are not treated with corticosteroids, or if such treatment is contraindicated.

Non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease: an open question.

AIMS: To review available data concerning the basic science and epidemiological-clinical evidence for an association of NAFLD and cardiovascular disease. DATA SYNTHESIS: Non-alcoholic fatty liver disease (NAFLD) defines alcohol-like hepatic histological lesions seen in the non-alcoholic, insulin resistant patient representing the hepatic counterpart of the metabolic syndrome. Along with insulin resistance, additional genetic, endocrine and vascular changes together with environmental stimuli--which are also involved in the pathogenesis of atherosclerosis--play a prominent role in the development and progression of NAFLD. Clinical and epidemiological studies seem to indicate that NAFLD is associated with an increased risk for cardiovascular disease but further studies are needed to confirm the available data. The mainstay of NAFLD treatment is based on the correction of the same metabolic changes that predispose to atherosclerosis. CONCLUSIONS: Non-invasive evaluation of risk for cardiovascular events is recommended in all individuals presenting with NAFLD and conversely, the presence of NAFLD should always be looked for in subjects with features belonging to the metabolic syndrome. Further studies are needed on the mechanisms linking fatty liver and vascular diseases.

Spontaneous rupture of the hepatic artery in a patient with type 1 neurofibromatosis treated by embolization: a case report.

We report the case of a 48-year-old man with neurofibromatosis presenting with sudden-onset abdominal pain, profound hypotension, and a drop in hemoglobin. CT scan demonstrated a massive hematoma within the right lobe of the liver with rupture into the peritoneal cavity. Angiography demonstrated diffuse abnormalities of the hepatic circulation with fusifom, ectatic, and stenotic segments. Acute extravasation from a peripheral branch of the right hepatic artery was identified and successfully embolized with subsequent hemodynamic stabilization of the patient. To the best of our knowledge this is the first case report of this kind in a patient with type I neurofibromatosis.

Genes or environment to determine alcoholic liver disease and non-alcoholic fatty liver disease.

While the vast majority of heavy drinkers and individuals with obesity, insulin resistance, and the metabolic syndrome will have steatosis, only a minority will ever develop steatohepatitis, fibrosis, and cirrhosis. Genetic and environmental risk factors for advanced alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) seem likely to include factors that influence the severity of steatosis and oxidative stress, the cytokine milieu, the magnitude of the immune response, and/or the severity of fibrosis. For ALD, the dose and pattern of alcohol intake, along with obesity are the most important environmental factors determining disease risk. For NAFLD, dietary saturated fat and antioxidant intake and small bowel bacterial overgrowth may play a role. Family studies and interethnic variations in susceptibility suggest that genetic factors are important in determining disease risk. For ALD, functional polymorphisms in the alcohol dehydrogenases and aldehyde dehydrogenase alcohol metabolising genes play a role in determining susceptibility in Oriental populations. No genetic associations with advanced NAFLD have been replicated in large studies. Preliminary data suggest that polymorphisms in the genes encoding microsomal triglyceride transfer protein, superoxide dismutase 2, the CD14 endotoxin receptor, TNF-alpha, transforming growth factor-beta, and angiotensinogen may be associated with steatohepatitis and/or fibrosis.

Popliteal artery branching patterns -- an angiographic study.

AIM: To analyse the variations in branching patterns of the popliteal artery and infrapopliteal vessels using angiography. MATERIALS AND METHODS: Femoral angiograms of 1037 lower limbs in 568 patients were examined to assess the popliteal artery branching pattern. Variations of the infrapopliteal vessels supplying the foot were assessed in 662 limbs from the same cohort of patients. RESULTS: Nine hundred and forty-one (90.7%) limbs had the usual branching pattern with anterior tibial artery (AT) arising first followed by the tibial-peroneal trunk (TPT), which then gives rise to the posterior tibial (PT) and peroneal (PR) arteries. Variations in popliteal branching pattern were seen in 96 (9.3%) limbs. The commonest variation is high origin of the AT in 47 (4.5%) limbs or trifurcation of the popliteal artery in 33 (3.2%) limbs with AT, PT and PR arising together with no true TPT. The course of AT with high origin either anterior or posterior to popliteus was almost equal [25 (2.4%) or 22 (2.1%) limbs, respectively]. Eleven limbs (1.1%) had high origin of PT and two (0.2%) had a high origin of the PR. Six hundred and fifty-five (99%) limbs had normal infrapopliteal vessels. Seven (1%) had hypoplasia-aplasia of the infrapopliteal vessels. Five (0.8%) limbs had a hypoplastic PT with the remaining 2 (0.2%) having either a hypoplastic-aplastic AT or hypoplasia-aplasia of both AT and PT. CONCLUSION: Variations in the branching of the popliteal artery occur in about 10% of patients. Knowledge of these variations is important because of the potential consequences for the management of peripheral vascular disease.

Mutant Bik expression mediated by the enhanced minimal topoisomerase IIalpha promoter selectively suppressed breast tumors in an animal model.

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we determined that topoisomerase IIalpha promoter is selectively activated in breast cancer cells. An element containing an inverted CCAAT box (ICB) was shown to be responsible for the breast cancer specificity. When the ICB-harboring topoisomerase IIalpha minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent proapoptotic gene, was shown to selectively kill breast cancer cells in vitro, and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs (such as heart) of CT90-BikDD-treated animals. The results indicate that liposomal CT90-BikDD is an effective systemic breast cancer-targeting gene therapy.

Polymorphisms in the T cell regulatory gene cytotoxic T lymphocyte antigen 4 influence the rate of acute rejection after liver transplantation.

BACKGROUND: The cytotoxic T lymphocyte antigen 4 (CTLA-4) gene encodes for a membrane bound (mCTLA-4) and a soluble (sCTLA-4) isoform, which are both involved in regulation of T cell function. The CTLA-4 +49A/G single nucleotide polymorphism (SNP) influences expression of mCTLA-4; +6230G/A SNP affects the production of sCTLA-4. AIM: To examine whether these functional SNPs influence the rate of rejection after liver transplantation. PATIENTS AND METHODS: Liver graft recipients (n = 483) were genotyped for both SNPs, and haplotypes were reconstructed. Association with rejection was tested by the log rank test using the Kaplan-Meier method with time to the first acute rejection episode as outcome. Multiple analysis of SNPs together with demographic factors was performed by Cox regression. RESULTS: Three haplotypes were observed in the cohort: +49A/+6230A, +49A/+6230G, and +49G/+6230G. The +49A/+6230G haplotype was significantly and dose dependently associated with acute rejection (p = 0.01). Of the demographic factors tested, only underlying liver disease was significantly associated with rejection. Adjusted for underlying liver disease, each additional +49A/+6230G haplotype allele resulted in a significantly higher risk of acute rejection (risk ratio 1.34 (95% confidence interval 1.04-1.72); p = 0.02). Patients who lacked this haplotype had the lowest, carriers an intermediate, and homozygotes the highest risk of acute rejection. CONCLUSION: The CTLA-4 +49A/+6230G haplotype, which encodes for normal mCTLA-4 expression but reduced sCTLA-4 production, is a co-dominant risk allele for acute rejection after clinical liver transplantation. This implies that even under immunosuppression, CTLA-4 is critically involved in the regulation of the human immune response to allogeneic grafts.

Analysis of factors predictive of mortality in alcoholic hepatitis and derivation and validation of the Glasgow alcoholic hepatitis score.

INTRODUCTION: Alcoholic hepatitis is associated with a high short term mortality. We aimed to identify those factors associated with mortality and define a simple score which would predict outcome in our population. METHODS: We identified 241 patients with alcoholic hepatitis. Clinical and laboratory data were recorded on the day of admission (day 1) and on days 6-9. Stepwise logistic regression was used to identify variables related to outcome at 28 days and 84 days after admission. These variables were included in the Glasgow alcoholic hepatitis score (GAHS) and its ability to predict outcome assessed. The GAHS was validated in a separate dataset of 195 patients. RESULTS: The GAHS was derived from five variables independently associated with outcome: age (p = 0.001) and, from day 1 results, serum bilirubin (p<0.001), blood urea (p = 0.019) and, from day 6-9 results, serum bilirubin (p<0.001), prothrombin time (p = 0.002), and peripheral blood white blood cell count (p = 0.001). The GAHS on day 1 had an overall accuracy of 81% when predicting 28 day outcome. In contrast, the modified discriminant function had an overall accuracy of 49%. Similar results were found using information at 6-9 days and when predicting 84 day outcome. The accuracy of the GAHS was confirmed by the validation study of 195 patients The GAHS was equally accurate irrespective of the use of the international normalised ratio or prothrombin time ratio, or if the diagnosis of alcoholic hepatitis was biopsy proven or on the basis of clinical assessment. CONCLUSIONS: Using variables associated with mortality we have derived and validated an accurate scoring system to assess outcome in alcoholic hepatitis. This score was able to identify patients at greatest risk of death throughout their admission.

Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis.

AIMS: Factors responsible for the progression of non-alcoholic fatty liver disease (NAFLD) to more severe liver injury are poorly understood. In the present study, we investigated the association between immune reactions triggered by oxidative stress and stage of NAFLD. METHODS: Titres of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) or arachidonic acid hydroperoxide (AAHP) and against oxidised cardiolipin (Ox-CL) were measured in 167 NAFLD patients with steatosis only (n = 79), steatohepatitis (n = 74), or steatosis plus cirrhosis (n = 14), and in 59 age and sex matched controls. RESULTS: Circulating IgG against lipid peroxidation products was significantly higher (p<0.001) in NAFLD patients than in controls. Oxidative stress dependent immune responses were not associated with obesity, type 2 diabetes, or with serum cholesterol, ferritin, or aminotransferase levels. Titres of lipid peroxidation related antibodies were also independent of the extent of steatosis and were similarly distributed in patients with and without necroinflammation. In contrast, the same antibodies were significantly increased in patients with advanced fibrosis or cirrhosis. Logistic regression analysis confirmed that anti-MDA antibodies were independently associated with progression of NALFD and that NAFLD patients with titres of anti-MDA-HSA antibodies above the control threshold value had a threefold (relative risk 2.82 (95% confidence interval 1.35-5.90); p = 0.007) higher risk of having advanced fibrosis/cirrhosis than patients whose antibody titres were within the control range. CONCLUSIONS: These results indicate that the presence of immune reactions triggered by oxidative stress can be an independent predictor of progression of NAFLD to advanced fibrosis.