Raquel Gonçalves

Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence:

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66u2009±u20090.69; TLR4: 3.11u2009±u20090.79; Pu2009<u20090.05) and cirrhosis (TLR2: 2.14u2009±u20090.5; TLR4: 1.74u2009±u20090.27; Pu2009<u20090.05) and decreased in hepatocarcinoma (TLR2: 0.48u2009±u20090.15; TLR4: 0.54u2009±u20090.10; Pu2009<u20090.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24u2009±u20090.79; COX-2: 2.47u2009±u20090.36; Pu2009<u20090.05) and cirrhosis (TNF-α: 1.73u2009±u20090.28; COX-2: 1.8u2009±u20090.35, Pu2009<u20090.05), whereas NF-κB mRNA was increased in hepatitis (2.42u2009±u20090.31; Pu2009<u20090.05) and unchanged in cirrhosis (1.34u2009±u20090.17; Pu2009=u20090.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63u2009±u20090.15; Pu2009<u20090.05) and maintained (at decreased levels) mRNA of NF-κB (0.52u2009±u20090.12) and TNF-α (0.52u2009±u20090.12; Pu2009<u20090.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence.

Phenotype–genotype profiles in Crohn's disease predicted by genetic markers in autophagy‐related genes (GOIA study II)

Background:About 70 loci are associated with susceptibility to Crohns disease (CD), particularly in pathways of innate immunity, autophagy, and pathogen recognition. Phenotype–genotype associations are inconsistent. Methods:CD susceptibility polymorphisms ATG16L1 rs2241880, ICAM1 rs5498, IL4 rs2070874, IL17F rs763780, IRGM rs13361189, ITLN1 rs2274910, LRRK2 rs11175593, and TLR4 rs4986790 were genotyped in a Portuguese population (511 CD patients, 626 controls) and assessed for association with CD clinical characteristics. Results:There is a significant association of CD with the single nucleotide polymorphisms (SNPs) in ATG16L1 (odds ratio [OR] 1.36 [1.15–1.60], P = 2.7 × 10−4 for allele G), IRGM (OR 1.56 [1.21–1.93], P = 3.9 × 10−4 for allele C), and ITLN1 (OR 1.55 [1.28–1.88], P = 4.9 × 10−6 for allele C). These SNPs are associated with ileal location (OR, respectively, 1.49, 1.52, and 1.70), ileocolonic location (OR, respectively, 1.31, 1.57, and 1.68), and involvement of the upper digestive tract (OR, respectively for ATG16L1 and IRGM, 1.96 and 1.95). The risk genotype GG in ATG16L1 is associated with patients who respond to steroids (OR 1.89), respond to immunosuppressants (OR 1.77), and to biologic therapy (OR 1.89). The SNPs in ITLN1 and IRGM are both associated with a positive response to biologic therapy. The risk for ileal, ileocolonic, and upper digestive tract locations increases with the number of risk alleles (OR for three alleles, respectively, 7.10, 3.54, and 12.07); the OR for positive response to biologic therapy is 3.66. Conclusions:A multilocus approach using autophagy-related genes provides insight into CD phenotype–genotype associations and genetic markers for predicting therapeutic responses.

Crohn's disease in a southern European country: Montreal classification and clinical activity

Background: Given the heterogeneous nature of Crohns disease (CD), our aim was to apply the Montreal Classification to a large cohort of Portuguese patients with CD in order to identify potential predictive regarding the need for medical and/or surgical treatment. Methods: A cross‐sectional study was used based on data from an on‐line registry of patients with CD. Results: Of the 1692 patients with 5 or more years of disease, 747 (44%) were male and 945 (56%) female. On multivariate analysis the A2 group was an independent risk factor of the need for steroids (odds ratio [OR] 1.6, 95% confidence interval [CI] 1.1–2.3) and the A1 and A2 groups for immunosuppressants (OR 2.2; CI 1.2–3.8; OR 1.4; CI 1.0–2.0, respectively). An L3+L34 and L4 location were risk factors for immunosuppression (OR 1.9; CI 1.5–2.4), whereas an L1 location was significantly associated with the need for abdominal surgery (P < 0.001). After 20 years of disease, less than 10% of patients persisted without steroids, immunosuppression, or surgery. The Montreal Classification allowed us to identify different groups of disease severity: A1 were more immunosuppressed without surgery, most of A2 patients were submitted to surgery, and 52% of L1+L14 patients were operated without immunosuppressants. Conclusions: Stratifying patients according to the Montreal Classification may prove useful in identifying different phenotypes with different therapies and severity. Most of our patients have severe disease. (Inflamm Bowel Dis 2009)

Ulcerative colitis in northern Portugal and Galicia in Spain

Background: Clinical and therapeutic patterns of ulcerative colitis (UC) are variable in different world regions. The purpose of this study was to examine two close independent southern European UC populations from 2 bordering countries and observe how demographic and clinical characteristics of patients can influence the severity of UC. Methods: A cross‐sectional study was conducted during a 15‐month period (September 2005 to December 2006) based on data of 2 Web registries of UC patients. Patients were stratified according to the Montreal Classification and disease severity was defined by the type of treatment taken. Results: A total of 1549 UC patients were included, 1008 (65%) from northern Portugal and 541 (35%) from Galicia (northwest Spain). A female predominance (57%) was observed in Portuguese patients (P < 0.001). The median age at diagnosis was 35 years and median years of disease was 7. The majority of patients (53%) were treated only with mesalamine, while 15% had taken immunosuppressant drugs, and 3% biologic treatment. Most patients in both groups were not at risk for aggressive therapy. Extensive colitis was a predictive risk factor for immunosuppression in northern Portugal and Galicia (odds ratio [OR] 2.737, 95% confidence interval [CI]: 1.846–4.058; OR 5.799, 95% CI: 3.433–9.795, respectively) and biologic treatment in Galicia (OR 6.329, 95% CI: 2.641–15.166). Younger patients presented a severe course at onset with more frequent use of immunosuppressors in both countries. Conclusions: In a large population of UC patients from two independent southern European countries, most patients did not require aggressive therapy, but extensive colitis was a clear risk factor for more severe disease. (Inflamm Bowel Dis 2010)

Inflammatory bowel disease: a patient's and caregiver's perspective.

The purpose of this study was to conduct a survey examining the impact of inflammatory bowel disease (IBD) on patients’ and their caregivers’ daily activities. Questionnaires were distributed to patients registered in the APDI (Portuguese Association for IBD) database and their respective caregivers in 2007. Of 422 patient respondents, 251 had Crohn’s disease (CD) and 171 had ulcerative colitis (UC), with the majority of patients being women (58.1%) and aged over 40xa0years (37.4%). The number of disease flares experienced by IBD patients was slightly higher for patients with CD than for patients with UC (2.64 vs. 2.34), and surgery was more often required in CD patients as compared to UC patients (42.4 vs. 7%). Sixty percent (60%) of patients reported having no problems with mobility, daily activities, or personal hygiene; however, over half of all patients experienced some pain and anxiety. Adult patients and children and adolescents respectively experienced time off work or school due to their disease but caregivers were not affected in this regard. The caregivers life (Nxa0=xa0324) was affected by anxiety, with the major concern reported as the risk of the patient developing cancer. Both IBD patients and caregivers thought that the provision of information on new drugs and contact time with a doctor would have the biggest impact on improving care. The symptoms and complications of IBD have a considerable impact on the lives of patients and their caregivers, and several actions could be taken to improve their care.

Estimating the prevalence of inflammatory bowel disease in Portugal using a pharmaco-epidemiological approach.

To estimate inflammatory bowel disease (IBD) prevalence in Portugal from 2003 to 2007, and to obtain disease, sex and age specific estimates.

Proactive therapeutic drug monitoring of infliximab: a comparative study of a new point‐of‐care quantitative test with two established ELISA assays

Therapeutic drug monitoring is a powerful strategy known to improve the clinical outcomes and to optimise the healthcare resources in the treatment of autoimmune diseases. Currently, most of the methods commercially available for the quantification of infliximab (IFX) are ELISA‐based, with a turnaround time of approximately 8 h, and delaying the target dosage adjustment to the following infusion.

Ulcerative colitis in a Southern European country: a national perspective

Background:The incidence, prevalence, and even the clinical behavior of ulcerative colitis (UC) are highly variable in different world regions. In previous studies, Portugal was reported as having a milder clinical behavior. The aim of this study was to apply the Montreal Classification in a large group of UC Portuguese patients in order to describe their clinical characteristics and evaluate variables potentially useful for outcome prediction. Methods:A cross‐sectional study based on data collected from a nationwide online registry was undertaken. Results:In all, 2863 patients with UC were included. Twenty‐one percent had ulcerative proctitis, 52% left‐sided colitis, and 28% extensive colitis. Sixty percent of patients had taken steroids, 14% immunosuppressors, 1% biologicals, and 4.5% were submitted to surgery. Patients with extensive colitis had more severe activity, needing more steroids, immunosuppressors, and surgery. At the time of diagnosis 61% were less than 40 years old and 5% less than 16. Younger patients also had a more aggressive initial course. Thirty‐eight percent of patients had only taken salicylates during the disease course and were characterized by a lower incidence of systemic symptoms at presentation (3.8% versus 8.8%, P < 0.001), fewer extraintestinal manifestations (7.7% versus 24.0%, P < 0.001), and a higher prevalence of proctitis (32.1% versus 10.0%). Conclusions:A more aggressive phenotype was found in extensive colitis and in the initial course of younger patients, with an increased need for steroids and immunosuppressors. In addition, a significant percentage of patients, particularly with proctitis, showed a milder clinical evolution and were maintained in remission only with salicylates. (Inflamm Bowel Dis 2009)

Anaemia in Patients with Inflammatory Bowel Disease - A Nationwide Cross-Sectional Study

Background: Anaemia is the most common complication in patients with inflammatory bowel disease (IBD). This study aims to assess the prevalence of anaemia in IBD patients and to know its characteristics with regard to the main IBD clinical features. Methods: An observational cross-sectional multicentre study was conducted. We included all patients who had an appointment at the 15 participating centres during the period of 1 month, and who met the following selection criteria: age ≥18, diagnosis of IBD. Disease activity was evaluated by Harvey-Bradshaw Index (HBI) for Crohns disease (CD), and by Simple Clinical Colitis Activity Index (SCCAI) for ulcerative colitis (UC). Results: One thousand three hundred and thirteen patients, were included: 54.8% female, mean age 42.8 (interquartile range (25th-75th): 31-53 years), 59% had a diagnosis of CD, 39% of UC and 2% IBD unclassified. The median follow-up since diagnosis was 7 years. The ongoing treatment was aminosalicylates (63.1%), corticosteroids (11.6%), immunomodulators (36.4%) and anti-tumour necrosis factor (27.3%). Anaemia was identified in 244 patients, representing a prevalence of 18.6% (95% CI 16.6-20.9). A majority of cases (90%) have mild/moderate anaemia (mean haemoglobin 11.3 ± 0.8 g/dl). Anaemia was significantly higher in females (p = 0.006), but there were no differences between CDs (19.1%) and UCs (17.7%; p = 0.688). Anaemia was more frequent in patients with active disease (HBI >4; SCCAI >2) than in those in clinical remission (33.6 vs. 15.6%, p < 0.001) and in patients on steroids (36.8%) vs. other treatments (p < 0.001). Only 47% of patients with anaemia were under any specific treatment (oral iron 67%; intravenous iron 41%). Conclusion: Anaemia was more frequent in patients with active disease and in those on corticosteroids. The treatment of anaemia still seems undervalued, whereas more than half of anaemic patients were not receiving any specific treatment and the use of oral iron prevails contrarily to current recommendations.

Detection of anti-infliximab antibodies is impacted by antibody titer, infliximab level and IgG4 antibodies: a systematic comparison of three different assays:

Background: There is scant information on the accuracy of different assays used to measure anti-infliximab antibodies (ADAs), especially in the presence of detectable infliximab (IFX). We thus aimed to evaluate and compare three different assays for the detection of IFX and ADAs and to clarify the impact of the presence of circulating IFX on the accuracy of the ADA assays. Methods: Blood samples from 79 ulcerative colitis (UC) patients treated with infliximab were assessed for IFX levels and ADAs using three different assays: an in-house assay and two commercial kits, Immundiagnostik and Theradiag. Sera samples with ADAs and undetectable levels of IFX were spiked with exogenous IFX and analyzed for ADAs. Results: The three assays showed 81–96% agreement for the measured IFX level. However, the in-house assay and Immundiagnostik assays detected ADAs in 34 out of 79 samples, whereas Theradiag only detected ADAs in 24 samples. Samples negative for ADAs with Theradiag, but ADA-positive in both the in-house and Immundiagnostik assays, were positive for IFX or IgG4 ADAs. In spiking experiments, a low concentration of exogenous IFX (5 µg/ml) hampered ADA detection with Theradiag in sera samples with ADA levels of between 3 and 10 µg/ml. In the Immundiagnostik assay detection interference was only observed at concentrations of exogenous IFX higher than 30 µg/ml. However, in samples with high levels of ADAs (>25 µg/ml) interference was only observed at IFX concentrations higher than 100 µg/ml in all three assays. Binary (IFX/ADA) stratification of the results showed that IFX+/ADA- and IFX-/ADAs+ were less influenced by the assay results than the double-positive (IFX+/ADAs+) and double-negative (IFX-/ADAs-) combination. Conclusions: All three methodologies are equally suitable for measuring IFX levels. However, erroneous therapeutic decisions may occur when patients show double-negative (IFX-/ADAs-) or double-positive (IFX+/ADAs+) status, since agreement between assays is significantly lower in these circumstances.