Wilson's Disease: A Prevalence Study in a Portuguese Population.

Introduction Wilson's disease (WD) is a rare and underdiagnosed genetic disorder caused by anomalous tissue copper deposition, and for which epidemiological studies, specifically in Portugal, are scarce. Objectives This study aimed to evaluate the prevalence and incidence of WD and provide a description of its main clinical and laboratory features. Methods A retrospective study was carried out, with a search between 1995 and 2015, of all patients with a minimum follow-up of three months and birth confirmed in the northern region of Portugal, with an estimated population of 3,689,682 inhabitants. Database collection was based on the Portuguese National Health Service's clinical coding system, relying on clinical data from 13 northern Portuguese hospitals, liver biopsy histology results, and hospital prescription records. Clinical and biochemical correlations were statistically assessed using chi-square, Mann-Whitney U, Friedman, and Wilcoxon tests. Results Over the 20-year period, a prevalence of 1:37.000 and an incidence of one per million person-year was found. A total of 94 patients were analyzed, with a slight male predominance (53%), the majority with the onset of clinical manifestations in pediatric age (56%), with a median age at diagnosis of 16.6 years (interquartile range of 12.3-20,.8 years). Most patients presented with predominant liver disease (54.8%), with more than a third with cirrhosis; mixed hepatic and neurological manifestations in 17.9%; and mainly neurological symptoms in 10.7% of the patients. Neurological impairment was strongly associated with delayed development of the manifestations of the disease (p = 0.001) and also a higher detection of Kayser-Fleischer rings (p < 0.001), present in 27.0% of the patients. Regarding therapy, penicillamine has been the most widely used, with adverse reactions reported in 24.8%. At six and 12 months after initiation of therapy, a significant decrease in liver enzymes was found (ALT: p = 0.002; AST: p = 0.002, respectively), but no significant reduction was observed in urinary copper excretion. Conclusion This was one of the first studies regarding WD prevalence in a Portuguese population, contributing to a better understanding of the epidemiology, diagnosis, and management of WD in the northern region of Portugal. WD should be considered in any individual with unexplained hepatic or neurological manifestations, and initial symptoms may manifest at an early age, even in children less than five years old. A high percentage of patients were identified in the early stages of the disease by asymptomatic elevation of transaminases. Following copper chelation therapy, cytolysis markers appear to be more sensitive indicators of treatment response.

Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis.

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB-/- and MTTP-/- organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Long-term outcome of patients with difficult-to-treat autoimmune hepatitis receiving mycophenolate mofetil.

BACKGROUND: Most patients with autoimmune hepatitis (AIH) respond to a combination of prednisolone and azathioprine. For patients who are intolerant or refractory to azathioprine, proposed alternative therapies are based on scarce data, limited to transplant centres and with short-term follow-up periods. OBJECTIVE: To evaluate the long-term efficacy and safety of MMF as a second-line therapy in patients with AIH managed at a tertiary non-transplant centre. METHODS: Retrospective analysis of a prospectively collated database identified AIH patients who received MMF from 2006 to 2015. Clinical, biochemical and immunological parameters were assessed at 3-, 6- and 12-months, and at last follow-up. Biochemical response (BR) was defined as improvement of transaminases, complete remission (CR) as normalisation of transaminases and IgG, while others were considered non-responders (NR). RESULTS: Eighteen out of 151 (12%) AIH patients received MMF. Nine received MMF due to azathioprine-intolerance (group 1), while nine due to refractory disease (group 2). In group 1, CR and BR was achieved in six (67%) and two (22%) patients respectively. In group 2, CR and BR was achieved in one (11%) and five (56%) patients respectively. Adverse events occurred in eight patients (44%), with one patient requiring drug discontinuation. After a medium follow-up of 78 (31-116) months, there was a significant decrease in transaminase levels, mirrored by decrease in prednisolone dose from 25 to 6.25 mg/day (P<0.05). CONCLUSION: Long-term therapy with MMF is safe and effective in AIH patients requiring second-line therapies, and these patients can be effectively managed at tertiary non-liver transplant centres.

Immediate effects of Pilates based therapeutic exercise on postural control of young individuals with non-specific low back pain: A randomized controlled trial.

OBJECTIVES: Low back pain affects the person's ability to keep balance, especially in challenging conditions. The purpose of this study was to determine the immediate effects of Pilates exercises on postural sway and dynamic balance of young individuals with non-specific low back pain. DESIGN: Controlled laboratory design. SETTINGS AND MAIN OUTCOME MEASURES: Forty-six participants with non-specific low back pain were randomized to a Pilates (n=23, 10 males; age: 21.8±3.2years) and a control group (n=23, 9 males; age: 22.8±3.6years). Postural sway was assessed with a force platform and dynamic balance with the Star Excursion Balance Test, before and after the intervention or rest period. To assess postural sway, participants stood still on an unstable surface set on the force plate for 90s, with eyes closed. INTERVENTION: The intervention lasted 20min and consisted on four Pilates exercises: single leg stretch (level 1), pelvic press (level 1), swimming (level 1) and kneeling opposite arm and leg reach. RESULTS: At baseline, no differences were found between groups. The Pilates group improved in all the postural sway values (area of CoP: 11.5±3.4 to 9.7±2.7cm2, p=0.002 and CoP velocity: 2.8±0.6 to 2.3±0.5cm/s, p<0.001) and in the Star Excursion Balance Test. Control group only improved in CoP velocity, however, this improvement was significantly inferior compared to the Pilates group. CONCLUSIONS: Pilates exercises immediately improved postural sway and dynamic balance in young adults with non-specific low back pain.

Polymicrobial Ventilator-Associated Pneumonia: Fighting In Vitro Candida albicans-Pseudomonas aeruginosa Biofilms with Antifungal-Antibacterial Combination Therapy.

The polymicrobial nature of ventilator-associated pneumonia (VAP) is now evident, with mixed bacterial-fungal biofilms colonizing the VAP endotracheal tube (ETT) surface. The microbial interplay within this infection may contribute for enhanced pathogenesis and exert impact towards antimicrobial therapy. Consequently, the high mortality/morbidity rates associated to VAP and the worldwide increase in antibiotic resistance has promoted the search for novel therapeutic strategies to fight VAP polymicrobial infections. Under this scope, this work aimed to assess the activity of mono- vs combinational antimicrobial therapy using one antibiotic (Polymyxin B; PolyB) and one antifungal (Amphotericin B; AmB) agent against polymicrobial biofilms of Pseudomonas aeruginosa and Candida albicans. The action of isolated antimicrobials was firstly evaluated in single- and polymicrobial cultures, with AmB being more effective against C. albicans and PolyB against P. aeruginosa. Mixed planktonic cultures required equal or higher antimicrobial concentrations. In biofilms, only PolyB at relatively high concentrations could reduce P. aeruginosa in both monospecies and polymicrobial populations, with C. albicans displaying only punctual disturbances. PolyB and AmB exhibited a synergistic effect against P. aeruginosa and C. albicans mixed planktonic cultures, but only high doses (256 mg L-1) of PolyB were able to eradicate polymicrobial biofilms, with P. aeruginosa showing loss of cultivability (but not viability) at 2 h post-treatment, whilst C. albicans only started to be inhibited after 14 h. In conclusion, combination therapy involving an antibiotic and an antifungal agent holds an attractive therapeutic option to treat severe bacterial-fungal polymicrobial infections. Nevertheless, optimization of antimicrobial doses and further clinical pharmacokinetics/pharmacodynamics and toxicodynamics studies underpinning the optimal use of these drugs are urgently required to improve therapy effectiveness and avoid reinfection.

Histological Outcomes and Predictive Value of Faecal Markers in Moderately to Severely Active Ulcerative Colitis Patients Receiving Infliximab.

BACKGROUND AND AIMS: Histological healing has emerged as a promising therapeutic goal in ulcerative colitis. This is especially important in the context of biological therapies. The objectives of the present study were to investigate the ability of infliximab to induce histological remission in ulcerative colitis [UC] patients and to explore the utility of faecal calprotectin and lactoferrin in predicting histological activity. METHODS: Multi-centre, single-cohort, open-label, 52-week trial including moderately to severely biological-naïve UC patients receiving intravenous infliximab [5mg/kg]. The primary outcome was the proportion of patients with histological remission [Geboes index ≤ 3.0] after 8 weeks of treatment, scored by two independent pathologists. RESULTS: Twenty patients were included. The rate of histological remission increased from 5% at baseline to 15% and 35% at Week 8 and Week 52, respectively. At Week 8, 40% of patients were in clinical remission [Mayo ≤ 2] and 45% achieved mucosal healing [Mayo endoscopy subscore 0-1]. At Week 52, 25% of patients had clinical, endoscopic and histological remission. Faecal calprotectin and lactoferrin showed the highest correlation with histological activity at Week 8 (area under the curve [AUC] 94%, p = 0.017; and 96%, p = 0.013, respectively) and both markers revealed an excellent positive predictive value for this outcome at this time point [100%, p = 0.017; and 94%, p = 0.013, respectively]. CONCLUSIONS: Infliximab was able to induce histological remission. There was a good agreement between histology and faecal biomarkers. Faecal calprotectin and lactoferrin were good predictors of histological remission. Our data support inclusion of histology as a treatment target complementary to endoscopy in clinical trials when evaluating therapeutic response in UC.

Kinesiology taping does not change fibularis longus latency time and postural sway.

BACKGROUND: Kinesiology tape seems to improve muscle force, although little is known regarding its effect on latency time and postural sway. OBJECTIVES: To examine the effects of kinesiology taping on fibularis longus latency time and postural sway in healthy subjects. METHODS: Thirty participants were equally randomized into three groups, two experimental groups receiving kinesiology tape (EG1, from origin to insertion; EG2, from insertion to origin) and a control group. Before and 20-min after the intervention, postural sway was assessed on a force platform and fibularis longus latency time was recorded with surface electromyography during a sudden inversion perturbation. RESULTS: At baseline, no differences were found between groups regarding age, anthropometrics variables, postural sway and fibularis longus latency time. In both experimental groups, the application of tape did not change postural sway and fibularis longus latency time (EG1: 93.7 ± 15.0 to 89.9 ± 15.6 ms; EG2, 81.24 ± 14.21 to 81.57 ± 16.64, p < 0.05). Also, no changes were observed in the control group. CONCLUSION: Kinesiology tape seems not to enhance fibularis longus reaction time and postural sway in young healthy subjects.

Misleading Gastrointestinal Symptoms: The Ongoing Story of Chronic Lead Intoxication.

Inorganic lead intoxication has emerged as an important and challenging clinical problem owing to increased awareness of lead and enhanced surveillance of exposed individuals. However, recognition may not be very difficult when there is an obvious history of exposure. Our interest began a few years ago when we could trace an outbreak, following a patient who was admitted with colickly abdominal pain, convulsions, and coma. After that, 16 more cases were identified and characterized. All patients recovered completely after adequate chelation therapy. Although the clinical picture of lead intoxication is pleomorphic, the increased awareness of gastroenterologists in this subject may possibly bring chronically complaining difficult patients to an earlier, unexpected, and fairly treatable disease.

Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology.

Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.

Antibiotic resistance of mixed biofilms in cystic fibrosis: impact of emerging microorganisms on treatment of infection.

Cystic fibrosis (CF) is a genetic disorder associated with multispecies infections where interactions between classical and newly identified bacteria might be crucial to understanding the persistent colonisation in CF lungs. This study investigated the interactions between two emerging species, Inquilinus limosus and Dolosigranulum pigrum, and the conventional CF pathogen Pseudomonas aeruginosa by evaluating the ability to develop biofilms of mixed populations and then studying their susceptibility patterns to eight different antimicrobials. Monospecies biofilms formed by I. limosus and D. pigrum produced significantly less biomass than P. aeruginosa and displayed greater sensitivity to antimicrobials. However, when in dual-species biofilms with P. aeruginosa, the emerging species I. limosus and D. pigrum were crucial in increasing tolerance of the overall consortia to most antibiotics, even without a change in the number of biofilm-encased cells. These results may suggest that revising these and other species interactions in CF might enable the development of more suitable and effective therapies in the future.

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice.

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.