Therapeutic monoclonal antibodies represent one of the fastest growing segments in the pharmaceutical market. The growth of the segment has necessitated development of new efficient and cost saving platforms for the preparation and analysis of early candidates for faster and better antibody selection and characterization. We report on a new integrated platform for automated harvesting of whole unclarified cell-culture broths, followed by in-line tandem affinity-capture, pH neutralization and size-exclusion chromatography of recombinant antibodies expressed transiently in mammalian human embryonic kidney 293T-cells at the 1-L scale. The system consists of two bench-top chromatography instruments connected to a central unit with eight disposable filtration devices used for loading and filtering the cell cultures. The staggered parallel multi-step configuration of the system allows unattended processing of eight samples in less than 24h. The system was validated with a random panel of 45 whole-cell culture broths containing recombinant antibodies in the early profiling phase. The results showed that the overall performances of the preparative automated system were higher compared to the conventional downstream process including manual harvesting and purification. The mean recovery of purified material from the culture-broth was 66.7%, representing a 20% increase compared to that of the manual process. Moreover, the automated process reduced by 3-fold the amount of residual aggregates in the purified antibody fractions, indicating that the automated system allows the cost-efficient and timely preparation of antibodies in the 20-200 mg range, and covers the requirements for early in vitro and in vivo profiling and formulation of these drug candidates. (C) 2015 The Authors. Published by Elsevier B.V.

Summary Background Patients with primary sclerosing cholangitis ( PSC ) and inflammatory bowel disease ( IBD ) have a high risk of developing colorectal cancer and dysplasia. Ursodeoxycholic acid ( UDCA ) has been suggested to have chemopreventive effects on the development of colorectal cancer and dysplasia but long‐term data and larger trials are lacking. Aim To evaluate the effect of high dose (17–23 mg/kg/day) UDCA on colorectal neoplasia in a cohort of patients with PSC and IBD . Methods From our previous 5‐year randomised controlled trial of UDCA vs. placebo in PSC , we performed a follow‐up of 98 patients with concomitant IBD from entry of the trial 1996–1997 until 2009 for development of colorectal cancer or dysplasia. Results The total follow‐up time was 760 person‐years. Dysplasia/cancer‐free survival was compared between placebo‐ ( n = 50) and UDCA ‐treated ( n = 48) patients. There was a similar frequency of dysplasia or cancer after 5 years between patients originally assigned to UDCA or placebo (13% vs. 16%) and no difference in dysplasia/cancer‐free survival ( P = 0.46, log rank test). At the end of 2009 no difference in cancer‐free survival was detected, 30% of the placebo patients compared with 27% of UDCA patients had developed colorectal cancer or dysplasia. Conclusions Long‐term high dose ursodeoxycholic acid does not prevent colorectal cancer or dysplasia in patients with primary sclerosing cholangitis ‐associated inflammatory bowel disease .

Objective. The information concerning the morbidity and mortality of hereditary hemochromatosis is based primarily on clinical cohorts of symptomatic patients. The major aim of this study was to analyze the long-term prognosis for Swedish patients with this condition, with respect to both clinical features and survival, in relation to the route by which the disease was detected. Patients and methods. 373 patients with hemochromatosis detected through routine health check-ups (n = 153), family screening (n = 44), symptoms of arthralgia (n = 23), investigation of other diseases/symptoms (n = 108) or signs of liver disease (n = 45) were monitored for a mean period of 11.9 ± 5.8 years. The degree of liver fibrosis and survival were analyzed. Results. Overall survival among these patients was not significantly different from that of a matched normal population. The patients diagnosed through health check-ups and family screening were detected at an earlier age and had the highest rate of survival. Liver biopsy at the time of diagnosis revealed cirrhosis in 9% of those detected through the health check-ups and 5% in the case of family screening, compared with 13% for the group with arthralgia, 17% for other diseases/symptoms and 42% for liver disease. Conclusion. Health check-ups and family screening allow detection of hereditary hemochromatosis at an earlier age and with less advanced liver fibrosis, although a few of these patients have already developed cirrhosis. Our study indicates that iron indices should be included in health check-ups, and if abnormal, should lead to further investigation.

Aimn-To study sampling variability ofpercutaneousliverbiopsyin primary scl-erosingcholangitis (PSC). Methods-Onehundredandtwelvebiopsyspecimens (that is, 56 pairs) from 44 patients with PSC, confirmed by chol- angiography, were evaluated blindly. Six different features, qualitative grading of fourotherfeatures and staging accordingto Ludwig wereassessed. Results-Quantitative sampling vari-ability was confined mainly to just one grade orstage, although 11%(six of 56) ofthe biopsyspecimenpairsdiffered bymorethan one stage (7% (one of 15) in pairs>2cm in length). Qualitative sampling variabilities were between 18and 71%.Ad- vanceddisease (stages 3 or 4) wasmissedin 40%(twooffive) ofthe biopsyspecimenswhilecirrhosis was missed in 37%.Conclusion-Paired liver biopsy speci- mensshouldbetakeninclinical studies of PSCusingliverhistology forevaluationor prognosis. (_J Clin Pathol 1995;48:933-935)Keywords: Sampling variability, primary sclerosingcholangitis. Liver biopsy is essential for the diagnosis andcontrol ofliver disease. The risk ofsampling

Summary Anti-microbial peptides are important effectors in innate immunity. In the gut they defend against pathogens, shape the commensal microbiota and probably control intestinal homeostasis. Ulcerative colitis (UC), but not Crohn's disease, shows increased expression of inducible β-defensins (hBD-2, hBD-3 and hBD-4) in colonic epithelial cells. Does inducible defensin production precede the chronic intestinal inflammation characteristic of UC, or is it a consequence of the T cell-driven chronic inflammation? The aim was to analyse defensin mRNA and protein expression in colonic epithelial cells in two colitis mouse models resembling UC, the interleukin (IL)-2−/− mouse and the dextran sulphate sodium (DSS)-induced colitis mouse. Defensin mRNA was assayed by in situ hybridization and quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR). Defensin peptide was assayed by immunohistochemistry. Mouse β-defensin 3 (mBD-3, orthologue to hBD-2) was up-regulated strongly in colonic epithelium of 15-week-old IL-2−/− mice and DSS-induced colitis mice with chronic bowel inflammation, but not in apparently healthy IL-2−/− 5-week-old mice, IL-2+/− 15-week-old mice or in acute stage DSS mice. Up-regulation was seen both at the mRNA- and at the protein level (only mBD-3 investigated). IL-17, but not several other cytokines, including interferon (IFN)-γ, induced mBD-3 mRNA expression in mouse colon carcinoma cells. The mRNA expression level of the constitutively expressed α-defensin, cryptdin-4, was up-regulated marginally in acute stage DSS-colitis mice and in IL-2−/− mice before signs of colitis. Inducible β-defensin expression in colonic epithelium is the consequence of the chronic bowel inflammation caused by activated T cells releasing cytokines including IL-17.

OBJECTIVES:Autoimmune hepatitis (AIH) is a liver disease which, if untreated, may lead to liver cirrhosis and hepatic failure. Limited data exist regarding factors predicting the long-term outcome. The aims of this study were to investigate symptoms at presentation, prognostic features, management and treatment in relation to long-term outcome of AIH.MATERIAL AND METHODS:A cohort of 473 Swedish patients with AIH was characterized regarding initial symptoms and signs, factors predicting death and future need for liver transplantation. Survival and causes of death were retrieved from Swedish national registers.RESULTS:At diagnosis, fatigue was a predominant symptom (69%), 47% of the patients were jaundiced and 30% had liver cirrhosis. Another 10% developed cirrhosis during follow-up. Markedly elevated alanine aminotransferase levels at presentation were correlated with a better outcome. A high international normalized ratio (INR) at diagnosis was the only risk factor predicting a need for later liver transplantation. Histological cirrhosis, decompensation and non-response to initial treatment were all factors that correlated with a worse outcome. Overall life expectancy was generally favourable. However, most deaths were liver-related, e.g. liver failure, shock and gastrointestinal bleeding.CONCLUSIONS:Cirrhosis at diagnosis, a non-response to initial immune-suppressive treatment or elevated INR values were associated with worse outcome and a need for later liver transplantation. In contrast, an acute hepatitis-like onset with intact synthetic capacity indicated a good response to treatment and favourable long-term prognosis. Lifetime maintenance therapy is most often required.

Aliment Pharmacol Ther 2010; 32: 1154–1162SummaryBackground Reliable epidemiological data for portal vein thrombosis are lacking.Aims To investigate the incidence, prevalence and survival rates for patients with portal vein thrombosis.Methods Retrospective multicentre study of all patients registered with the diagnosis of portal vein thrombosis between 1995 and 2004.Results A total of 173 patients (median age 57 years, 93 men) with portal vein thrombosis were identified and followed up for a median of 2.5 years (range 0–9.7). The mean age‐standardized incidence and prevalence rates were 0.7 per 100 000 per year and 3.7 per 100 000 inhabitants, respectively. Liver disease was present in 70 patients (40%), malignancy in 27%, thrombophilic factors in 22% and myeloproliferative disorders in 11%. Two or more risk factors were identified in 80 patients (46%). At diagnosis, 65% were put on anticoagulant therapy. Thrombolysis, TIPS, surgical shunting and liver transplantation were performed in 6, 3, 2 and 8 patients, respectively. The overall survival at 1 year and 5 years was 69% and 54%. In the absence of malignancy and cirrhosis, the survival was 92% and 76%, respectively.Conclusions The incidence and prevalence rates of portal vein thrombosis were 0.7 per 100 000 inhabitants per year and 3.7 per 100 000 inhabitants, respectively. Concurrent prothrombotic risk factors are common. The prognosis is variable and highly dependent on underlying disease.

Autoimmune hepatitis (AIH) is a rare disease with an incidence of around 1 / 100,000 inhabitants, and a prevalence rate of 16 / 100,000. As many as 30 % of AIH-patients already have liver cirrhosis ...

BACKGROUND:Conditions exhibiting features of two different autoimmune liver diseases are designated overlap syndromes. Variant forms display some, but not all, characteristics of a distinct autoimmune liver disease. We describe transitions over time between variant forms of PBC, i.e. AMA-negative PBC, autoimmune hepatitis (AIH)-PBC overlap and autoimmune cholangitis (AIC) in a large cohort of PBC patients in Sweden. METHODS:We retrieved all patients with variant forms of PBC in six university hospitals in Sweden, covering 60% of the Swedish population. The diagnosis of PBC and its variants was based on laboratory findings and compatible histological features. The revised autoimmune hepatitis scoring system proposed by the International Autoimmune Hepatitis Group was used to establish the diagnosis of AIH. RESULTS:In a population of 800 patients with PBC, we identified 35 (5%) variant forms; 25 patients with AIH-PBC overlap, 8 with AIC and 2 with AMA-negative PBC at the time of our study. The initial diagnoses were PBC (3 patients), AIH (3), AIH-PBC overlap (16), AIC (8) and AMA-negative PBC with (1) or without (4) concomitant AIH. The median follow-up was 125 (41-360) months. Immunosuppression and ursodeoxycholic acid induced a complete or good regression of increased aminotransferases in about half of the patients who were given one or both of these treatments. CONCLUSIONS:Variant forms of PBC are seen in approximately 5% of PBC patients in Sweden. Transition between different forms may occur, emphasizing the value of repeat biopsies, but established overlapping AIH-PBC seems to be stable over time.

EPIDEMIOLOGY, DIAGNOSIS, MANAGEMENT) S303and resolution were established by placing the coil between a standard phantom and cuvettes containing resolution test samples.The micro-coil was located at the magnet isocentre, parallel to the magnet bore.3D GRE sequences containing 28 slices each 1.2 mm thick were used.Imaging was then carried out using meat models with the coil inserted into the biliary tree and liver parenchyma.Results: Scanning microfabricated test samples produced images showing entire cuvettes (1 cm deep) with 1 mm resolution test bars clearly visible.Imaging of porcine biliary tree revealed anatomical detail to a depth of more than 1.5 cm, 360 o around the coil, along 3.2 cm of its length.Branching bile ducts and blood vessels can be visualised clearly in the images presented.Conclusion: This MR probe can reliably produce images of a resolution of <1 mm and demonstrate anatomical detail in meat models.Work is underway to increase the useful length of the probe and on a clinical trial of the device.

Background: Overlap syndrome is a term used for overlapping features of autoimmune hepatitis and primary sclerosing cholangitis or primary biliary cirrhosis and for autoimmune cholangitis. We describe a high prevalence of small duct primary sclerosing cholangitis among patients with overlapping autoimmune hepatitis and primary sclerosing cholangitis.Methods: We sought to retrieve all patients with overlap syndrome between primary sclerosing cholangitis and autoimmune hepatitis in six university hospitals in Sweden. The revised autoimmune hepatitis scoring system proposed by the International Autoimmune Hepatitis Group was used to establish the diagnosis autoimmune hepatitis. Endoscopic retrograde cholangiography and/or magnetic resonance cholangiography were used to separate the primary sclerosing cholangitis cases diagnosed through liver biopsy into small and large primary sclerosing cholangitis. A histologocial diagnosis compatible with both autoimmune hepatitis and primary sclerosing cholangitis was required for inclusion.Results: 26 patients fulfilled our criteria for histological overlap of autoimmune hepatitis and primary sclerosing cholangitis, 7 (27%) of which had small duct primary sclerosing cholangitis. The reliability of the diagnosis small duct primary sclerosing cholangitis was supported by a very close similarity between small and large duct primary sclerosing cholangitis patients in clinical and laboratory data, and by a poor response to immunosuppressive therapy in the small duct primary sclerosing cholangitis patients. Patients with large duct overlap syndrome had a good response to immunosuppressive therapy. In both groups, our limited experience from ursodeoxycholic acid was largely poor.Conclusions: Small duct primary sclerosing cholangitis is prevalent in the overlap syndrome between autoimmune hepatitis and primary sclerosing cholangitis. (C) 2008 European Federation of Internal Medicine. Published by Elsevier B.V

Background/Aims: Autoimmune Hepatitis (AIH) is a liver disease which may lead to liver cirrhosis. Cirrhosis is a well-known risk factor for hepatocellular cancer. Lymphoma is a disease, where immune modulating drugs as well as the autoimmune disease itself may contribute to the elevated risk. The aim was to investigate the risks of malignancies in a large cohort of AIH patients.Methods: Four hundred and seventy-three patients with AIH were matched to the Swedish national cancer register as well as to the death cause register.Results: We found an overall higher risk of malignancies in the cohort of A I H patients from the date of diagnosis with a SIR of 1.51 (95% CI 1.10-2.03). SIR in the subpopulation of well defined catchment areas and complete case finding was 23.28 (95% CI 7.5-54.34) for HCC. Lymphomas were found a SIR of 13.09 (95% CI 4.22-30.56).Conclusions: There was an overall increased risk of malignancies in a cohort of AIH patients, which manly was caused by hepatobiliary cancers. However, the true risk of HCC in an AIH cirrhotic cohort has yet to be investigated. A significantly higher risk of lymphomas was also found, but no clear cut association to the use of immune modulators. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Regulatory T cells seem to play a central role in maintaining immune tolerance in the gut mucosa. Previously we have shown that interleukin (IL)-10 is produced at high levels in the inflamed colonic tissue of ulcerative colitis (UC) patients. The cellular source was CD4(+) T cells, suggesting local activation of regulatory T cells. The present study was performed to determine whether the frequency of regulatory T cells is increased in UC colon and whether they are present in the basal lymphoid aggregates, the prominent microanatomical structure in UC colon. Colonic tissue specimens from UC and control patients were analysed for frequencies of lamina propria lymphocytes expressing the regulatory T cell markers forkhead box protein 3 (FoxP3), CD25 and glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) as well as CD28, CD4 and CD3 by using marker specific reagents in immunomorphometry. Two-colour immunohistochemistry was used for detection of CD25/IL-10, FoxP3/IL-10 and CD25/FoxP3 double-positive cells. GITR(+) and FoxP3(+) cells were present in normal colon mucosa, although at a relatively low frequency, and were located preferentially within the solitary follicles. UC was associated with significantly increased frequencies of CD25(+), GITR(+) and FoxP3(+) lamina propria lymphocytes both within the basal lymphoid aggregates and in the lamina propria outside. Many of the CD25(+) cells co-expressed FoxP3 as well as IL-10, suggesting that these are indeed IL-10 secreting regulatory T cells, activated in an attempt to counteract the inflammation. Increased frequency of regulatory T cell subtypes seems insufficient to control the disease activity in UC.

BACKGROUND & AIMS:The importance of genetic factors for the development of primary sclerosing cholangitis (PSC) is incompletely understood. This study assessed the risk of PSC and inflammatory bowel disease (IBD) among first-degree relatives of patients with PSC, compared with the first-degree relatives of a cohort without PSC.METHODS:Subjects from the national Swedish cohort of PSC patients (n = 678) were matched for date of birth, sex, and region to up to 10 subjects without a diagnosis of PSC (n = 6347). Linkage through general population registers identified first-degree relatives of subjects in both the PSC and comparison cohorts (n = 34,092). Diagnoses among first-degree relatives were identified by using the Inpatient Register.RESULTS:The risk of cholangitis was statistically significantly increased in offspring, siblings, and parents of the PSC patient cohort, compared with relatives of the comparison cohort, with the hazard ratios and 95% confidence intervals, 11.5 (1.6-84.4), 11.1 (3.3-37.8), and 2.3 (0.9-6.1), respectively. The hazard ratios for ulcerative colitis (UC) among first-degree relatives of all PSC patients was 3.3 (2.3-4.9) and for Crohn's disease 1.4 (0.8-2.5). The risk of UC for relatives of PSC patients without IBD was also increased, 7.4 (2.9-18.9).CONCLUSIONS:First-degree relatives of patients with PSC run an increased risk of PSC, indicating the importance of genetic factors in the etiology of PSC. First-degree relatives of PSC patients without IBD are also at an increased risk of UC, which might indicate shared genetic susceptibility factors for PSC and UC.

OBJECTIVE:Autoimmune hepatitis (AIH) is a chronic liver disease, which if untreated can lead to cirrhosis and hepatic failure. The aim of the study was to investigate the incidence, prevalence, diagnostic tradition and clinical initial presentation of AIH. MATERIAL AND METHODS:Analyses were performed in 473 patients identified as having probable or definite AIH. RESULTS:The incidence of AIH was 0.85/100,000 (95% CI 0.69-1.01) inhabitants, which is somewhat lower than reported previously. The point prevalence amounted to 10.7/100,000 (95% CI 8.8-13.1), and 76% of the cases were females. The age-related incidence curve was bimodal but men were found to have only one incidence peak in the late teens, whereas women had a peak after menopause. AIH was presented as a spectrum of clinical settings from detected "en passant" to acute liver failure. Almost 30% of patients already had liver cirrhosis at diagnosis. Autoantibodies indicative of AIH type 1 were found in 79% of cases. Other concomitant autoimmune diseases were frequently found (49%). CONCLUSIONS:The incidence and prevalence figures confirm that AIH is a fairly uncommon disease in the Swedish population. Symptoms at presentation were unspecific, but almost half of the patients were jaundiced, with around 30% having liver cirrhosis. The majority of Swedish AIH patients had AIH type 1.

BACKGROUND:The exact incidence and prevalence of Budd-Chiari syndrome (BCS) is unknown in the general population. Published reports differ in terms of the clinical characteristics, effects of therapy and survival. AIMS:To investigate the epidemiology, clinical presentation and survival in patients with BCS. METHODS:Retrospective multicentre study in Sweden reviewing the medical records of all patients with BCS 1986-2003, identified from the computerised diagnosis database of 11 hospitals, including all university hospitals and liver transplantation centres. RESULTS:Forty-three patients with BCS were identified, of whom nine (21%) had concomitant portal vein thrombosis. The mean age-standardised incidence and prevalence rates in 1990-2001 were calculated to be 0.8 per million per year and 1.4 per million inhabitants respectively. Myeloproliferative disorders (38%), thrombophilic factors (31%) and oral contraceptives (30%) were common aetiological factors. Two or more risk factors were present in 44%. In 23%, no risk factor was evident. The median follow-up time was 2.7 years. Seventy-two percent were on anticoagulant therapy during follow-up. Transjugular intrahepatic portosystemic shunting, surgical shunting procedures and liver transplantation were performed in 4, 6 and 18 patients respectively. Nineteen patients died. The overall transplantation-free survival at 1, 5 and 10 years was 47, 28 and 17% respectively. CONCLUSIONS:Budd-Chiari syndrome is a rare disorder; the mean age-standardised incidence and prevalence rates in Sweden in 1990-2001 were calculated to be 0.8 per million per year and 1.4 per million inhabitants respectively. The presence of a myeloproliferative disorder was a common aetiological factor in our cohort and about half of the patients had a multifactorial aetiology. The transplantation-free survival was poor.