João-Bruno Soares

Ghrelin, des-acyl ghrelin and obestatin: Three pieces of the same puzzle

The major active product of ghrelin gene is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, called simply ghrelin. Ghrelin has a multiplicity of physiological functions, affecting GH release, food intake, energy and glucose homeostasis, gastrointestinal, cardiovascular, pulmonary and immune function, cell proliferation and differentiation and bone physiology. Nevertheless, recent developments have shown that ghrelin gene can generate various bioactive molecules besides ghrelin, mainly des-acyl ghrelin and obestatin, obtained from alternative splicing or from extensive post-translational modification. Although their receptors have not yet been identified, they have already proven to be active, having intriguingly subtle but opposite physiological actions to ghrelin. This suggests the existence of a novel endocrine system with multiple effector elements which not only may have opposite actions but may regulate the action of each other. In this review, we summarize the steps which lead to the production of the different ghrelin gene products and examine the most significant differences between them in terms of structure and actions.

The role of lipopolysaccharide/toll-like receptor 4 signaling in chronic liver diseases.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor that functions as lipopolysaccharide (LPS) sensor and whose activation results in the production of several pro-inflammatory, antiviral, and anti-bacterial cytokines. TLR4 is expressed in several cells of healthy liver. Despite the constant confrontation of hepatic TLR4 with gut-derived LPS, the normal liver does not show signs of inflammation due to its low expression of TLR4 and ability to modulate TLR4 signaling. Nevertheless, there is accumulating evidence that altered LPS/TLR4 signaling is a key player in the pathogenesis of many chronic liver diseases (CLD). In this review, we first describe TLR4 structure, ligands, and signaling. Later, we review liver expression of TLR4 and discuss the role of LPS/TLR4 signaling in the pathogenesis of CLD such as alcoholic liver disease, nonalcoholic fatty liver disease, chronic hepatitis C, chronic hepatitis B, primary sclerosing cholangitis, primary biliary cirrhosis, hepatic fibrosis, and hepatocarcinoma.

Inotropic and lusitropic effects of ghrelin and their modulation by the endocardial endothelium, NO, prostaglandins, GHS-R1a and KCa channels.

Contractile effects of ghrelin (10(-9) to 10(-6) M) were tested in rat papillary muscles of normal (n = 50) and hypertrophic (n = 16) right ventricles (RV). RV hypertrophy was induced by pulmonary hypertension using monocrotaline. In normal muscles, ghrelin was added either alone (n = 9) or after pre-treatment with indomethacin (cycloxygenase inhibitor, 10(-5) M; n = 10), L-nitro-L-arginin (NO synthase inhibitor, 10(-4) M; n = 9), D-Lys(3)-GHRP-6 (GHS-R1a antagonist; 10(-4) M; n = 8) or apamin+charybdotoxin (KCa channels blockers; 10(-6) M, n =7 ), as well as after damaging the endocardial endothelium (n = 7). In hypertrophic muscles, ghrelin was added either alone (n = 9) or after pre-treatment with apamin+charybdotoxin (10(-6 M, n=7). Ghrelin concentration-dependently decreased active tension (AT) and maximal velocity of tension rise (negative inotropic effect), as well as, maximal velocity of tension decay (negative lusitropic effect) and time to AT (onset of relaxation). These effects were maximal at 10(-6) M, similar in normal and hypertrophic muscles and were significantly altered only by apamin+charybdotoxin, indomethacin and L-nitro-L-arginin. Apamin+charybdotoxin attenuated the negative inotropic effect, while indomethacin and L-nitro-L-arginin, respectively, blunted and exacerbated the premature onset of relaxation. In conclusion, ghrelin induces negative inotropic and lusitropic effects and an earlier onset of relaxation in normal and hypertrophic myocardium, which are independent of GHS-R1a, since they were not affected by D-Lys(3)-GHRP-6. The negative inotropic effect is partly mediated by KCa channels, while the earlier onset of relaxation is modulated by prostaglandins and NO.

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: 2.66 ± 0.69; TLR4: 3.11 ± 0.79; P < 0.05) and cirrhosis (TLR2: 2.14 ± 0.5; TLR4: 1.74 ± 0.27; P < 0.05) and decreased in hepatocarcinoma (TLR2: 0.48 ± 0.15; TLR4: 0.54 ± 0.10; P < 0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24 ± 0.79; COX-2: 2.47 ± 0.36; P < 0.05) and cirrhosis (TNF-α: 1.73 ± 0.28; COX-2: 1.8 ± 0.35, P < 0.05), whereas NF-κB mRNA was increased in hepatitis (2.42 ± 0.31; P < 0.05) and unchanged in cirrhosis (1.34 ± 0.17; P = 0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63 ± 0.15; P < 0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52 ± 0.12) and TNF-α (0.52 ± 0.12; P < 0.05, 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.

Ghrelin and ghrelin receptor inhibitors: agents in the treatment of obesity.

Background: Current medical treatment of obesity is highly ineffective. Soon after its discovery as the endogenous ligand for the growth hormone secretagogue-receptor (GHS-R), ghrelin was shown to stimulate food intake (including in humans) and promote body weight gain and adipogenesis. Objectives: This review discusses the role of the ghrelin/GHS-R pathway in energy homeostasis regulation and its role as a novel molecular target for the treatment of obesity. Methods: Medline was searched for relevant articles published in English. Results/conclusion: A large series of animal studies shows that inhibition of the ghrelin/GHS-R pathway reduces food intake, body weight and adiposity, through reduction of appetite and augmentation of energy expenditure and fat catabolism. This suggests that inhibition of this novel pathway may be used to treat/prevent obesity and its complications.

Reprocessing practice in digestive endoscopy units of district hospitals: results of a Portuguese National Survey.

Background and aim An inadequate disinfection of endoscopes and associated accessories can result in the transmission of infections to patients. The aim of this study was to access reprocessing practice in the endoscopy units of Portuguese district general hospitals. Methods An anonymous questionnaire on cleaning and disinfection methods was sent to all endoscopy units of Portuguese district general hospitals. Results A total of 25 units responded (93%). All endoscopy units performed manual cleaning (including brushing of accessible channels) before disinfection. Automated endoscope reprocessing machines were available in all units. Manual disinfection was performed in only one unit. In 48% of the surveyed units, endoscopes were systematically disinfected before each session, whereas in 16% this was performed only occasionally. The most commonly used disinfectant was peracetic acid (32%). Disposable papillotomes, biopsy forceps, and polipectomy snares were used in nine (36%), six (24%), and 14 (56%) units, respectively. Disposable papillotomes, forceps, and snares were reused in three (12%), two (8%), and three (12%) units, respectively, always after sterilization. Most units did not perform regular evaluation of reprocessing staff competence (60%), regular microbiological inspection (56%), or registry of reprocessing (56%). Conclusion The data collected suggest that there is a good compliance with standard guidelines. Nevertheless, there is still room for improvement mainly in quality assurance.

Interobserver agreement of EUS elastography in the evaluation of solid pancreatic lesions

Background and Objectives: Previous reports assessing the reproducibility of endoscopic ultrasound elastography (EUS-E) in evaluation of solid pancreatic lesions (SPL) involved only experienced endosonographers. We aimed to assess the interobserver agreement (IOA) of EUS-E in the evaluation of SPL by endoscopists with different levels of experience in EUS and EUS-E. Materials and Methods: A cross-sectional observational multicenter study was designed and included 11 endoscopists who were divided into four groups: Group A (long experience in EUS and EUS-E); Group B (short experience in EUS and EUS-E); Group C (long experience in EUS and no experience in EUS-E); and Group D (no experience in EUS or EUS-E). The observers independently classified the patterns of 60 video sequences of EUS-E, after a 20-min training session. For each group, we calculated IOA (kappa statistic, κ) of EUS-E and the diagnostic accuracy of EUS-E for pancreatic malignancy, by comparing the pattern of EUS-E indicative of malignancy (heterogeneous or homogenous blue) with the final diagnosis. Results: The overall IOA was moderate (κ = 0.42; 95% confidence interval (CI) 0.33-0.52). The IOA of Group A (κ = 0.80; 95% CI 0.65-1.00) was significantly higher than that of Groups B (κ = 0.54; 95%CI 0.40-0.71), C (κ = 0.54; 95%CI 0.39-0.68), and D (κ = 0.28; 95%CI 0.14-0.40). IOA of Groups B and C was not significantly different, but it was significantly higher than that of Group D. The diagnostic accuracy of Group A (area under the curve under summary receiver operating characteristic (AUROC) = 0.83; 95%CI 0.75-0.90) was not significantly different from that of Group B (AUROC = 0.77; 95%CI 0.71-0.83), but it was significantly higher than that of Groups C (AUROC = 0.74; 95%CI 0.67-0.81) and D (AUROC = 0.74; 95%CI 0.67-0.81). No significant difference was seen between Groups B, C, and D for diagnostic accuracy. Conclusion: EUS-E is reproducible in the evaluation of SPL, even between endoscopists with no or limited experience in EUS and/or EUS-E. Reproducibility and diagnostic accuracy increase with experience in EUS and EUS-E.

Clinical performance of an infliximab rapid quantification assay

Background: Therapeutic drug monitoring (TDM)-based algorithms can be used to guide infliximab (IFX) adjustments in inflammatory bowel disease (IBD) patients. This study aimed to explore a rapid IFX-quantification test from a clinical perspective. Methods: This manuscript describes a prospective cohort study involving 110 ulcerative colitis (UC) patients on the maintenance phase of IFX. IFX trough levels were quantified using a rapid quantification assay and a commonly-used reference kit. Results: Irrespective of the assay used to measure IFX, its through levels were statistically different between patients with and without endoscopic remission (Mayo endoscopic score = 0), as well as between patients stratified by their faecal calprotectin (FC) levels. Despite the fact that the two methods correlated well with each other [Spearman’s rank correlation coefficient = 0.843, p < 0.001; intraclass correlation coefficients = 0.857, 95% confidence interval (CI): 0.791–0.903], there was a discernible systematic variation; values obtained with the reference kit were on average 2.62 units higher than those obtained with the rapid assay. Notwithstanding, 3 µg/ml was shown to be an acceptable cut-off to assess endoscopic status and inflammatory burden levels using both assays. The percentage of patients that had a positive outcome when the IFX concentration measured by the rapid assay ranked above 3 µg/ml was 88% both for a Mayo endoscopic score ⩽ 1 and for an FC concentration <250 µg/g. Conclusions: Based on this study, we concluded that using the rapid IFX assessment system with a 3 µg/ml threshold is a reliable alternative to the time-consuming enzyme-linked immunosorbent assays in patients on the maintenance phase of IFX.

Interobserver agreement of contrast-enhanced harmonic endoscopic ultrasonography in the evaluation of solid pancreatic lesions

Background and study aims: Previous reports assessing the reproducibility of contrast-enhanced harmonic endoscopic ultrasonography (CH-EUS) in the evaluation of solid pancreatic lesions (SPLs) involved mainly experienced endosonographers. We aimed to assess the interobserver agreement (IOA) of CH-EUS in the evaluation of SPLs by endoscopists with different levels of experience in EUS and CH-EUS. Participants and methods: A cross-sectional observational multicenter study was designed and included 11 endoscopists who were divided into four groups according to their experience in EUS and CH-EUS: group A (long experience in EUS and CH-EUS); group B (short experience in EUS and CH-EUS); group C (long experience in EUS and no experience in CH-EUS); and group D (no experience in EUS or CH-EUS). The observers independently classified the patterns of 60 CH-EUS video sequences of 60 SPLs after a 20-minute training session. For each group, we calculated the IOA (kappa statistic, κ) of CH-EUS and the accuracy of CH-EUS for the diagnosis of pancreatic adenocarcinoma by comparing the pattern of CH-EUS indicative of pancreatic adenocarcinoma (hypo-enhanced contrast pattern) with the final diagnosis. Results: The overall IOA for CH-EUS was fair (κ = 0.32; 95 %CI 0.22 – 0.41). Group A (κ = 0.63; 95 %CI 0.45 – 0.85) had the highest IOA, followed by group C (κ = 0.54; 95 %CI 0.39 – 0.71), group B (κ = 0.38; 95 %CI 0.22 – 0.55), and group D (κ = 0.21; 95 %CI 0.07 – 0.36). The IOA of groups A and C was significantly higher than that of group D. No significant difference was seen between groups A, B, and C or between groups B and D in terms of IOA. Group A (area under the curve under summary receiver operating characteristic [AUROC] = 0.67; 95 %CI 0.58 – 0.75) had the highest accuracy for the diagnosis of pancreatic adenocarcinoma, followed by group C (AUROC = 0.58; 95 %CI 0.50 – 0.65), group B (AUROC = 0.55; 95 %CI 0.48 – 0.63), and group D (AUROC = 0.51; 95 %CI 0.43 – 0.58). The diagnostic accuracy of group A was not significantly different from that of group C, but it was significantly higher than that of groups B and D. No significant difference was seen between groups B, C, and D in terms of diagnostic accuracy. Conclusions: CH-EUS is reproducible in the evaluation of SPLs, even between endoscopists with no or limited experience in EUS and/or CH-EUS. Long experience in EUS is a major contributor to the IOA and diagnostic accuracy of CH-EUS.

Efficacy and tolerability of culture-guided treatment for Helicobacter pylori infection

Objective The aim of this study was to evaluate the efficacy/tolerability of a culture-guided approach in the eradication of Helicobacter pylori and identify factors associated with antibiotic resistance/treatment failure. Patients and methods This retrospective single-center study included patients who underwent culture-guided treatment for H. pylori infection, after two ineffective eradication attempts, between October 2012 and December 2016. We assessed the following demographic and clinical data of the patients: sex, age, BMI, alcohol and tobacco consumption, history of dyspepsia, peptic ulceration and first-degree relatives with gastric cancer, antibiotic susceptibility results, treatment composition, tolerability, and success. The treatment success was confirmed by a monoclonal stool antigen test. Results Culture-guided treatment was performed in 42 patients (57% women, mean age±SD: 48.9±11.4 years). The rates of antibiotic resistance were as follows: clarithromycin 86%, metronidazole 67%; levofloxacin 52%, tetracycline 2%, and amoxicillin and rifampicin 0%. Double resistance to clarithromycin and metronidazole was found in 59.5% of the patients. Most patients showed resistance to less than three antibiotics, but 31% were resistant to three or more. Intention-to-treat and per-protocol eradication rates were 59.5 and 61.5%. Adverse events occurred in 15 (35.7%) patients, but only two (4.8%) patients did not complete treatment because of adverse events. Only age more than 50 years was associated with resistance to three or more antibiotics. Having a first-degree relative with gastric cancer was associated with treatment failure and having a BMI of at least 25 kg/m2 protected from failure. Conclusion Third-line culture-guided treatment often fails to eradicate H. pylori infection. We need to find factors other than in-vitro antibiotic resistance to explain these suboptimal results.