Design and Proof of Concept of a Web-Based Questionnaire to Identify Patients at Risk for HIV and HCV Infection.

Despite remarkable improvement in the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) care continuum, the rate of late diagnosis of HIV and HCV in high-income countries remains unacceptably high. Testing relies mainly on primary care physicians' identification of risk factors. We aimed to adapt an analogic to an online questionnaire to help HIV and HCV screening and perform a pilot study to assess its accuracy and acceptability. We used the Delphi method to adapt a previously validated analogical questionnaire to a user-friendly online tool. It aimed to identify participants who should be screened for HIV or HCV and those who should be referred for pre-exposure prophylaxis (PrEP). We then designed a proof-of-concept pilot study from July to October 2022 to test its feasibility and suitability for use on a larger scale and to assess its accuracy in identifying patients at risk for HIV or HCV or with indication for PrEP. The final questionnaire consisted of 37 questions. A total of 142 participants provided informed consent, and 102 completed the questionnaire: 41 random patients recruited at the primary care level, 10 participants recently diagnosed with HIV, 20 participants with HIV on follow-up, 21 participants from the PrEP program, and 10 patients diagnosed with HCV. The tool adequately indicated the need for testing in more than 98% of participants with confirmed HIV/HCV infections or in the PrEP program. Furthermore, it adequately assessed PrEP referral in 94% of participants already on PrEP or with known HIV infection. Participants were highly satisfied with the tool, and 98% of them recommended its use. A self-administered web-based tool to identify patients who should be tested for HIV or HCV or referred to PrEP could simplify patient selection and help reduce late diagnosis.

Long-term outcome and risk stratification in compensated advanced chronic liver disease after HCV-cure.

BACKGROUND AND AIMS: Around 750,000 patients per year will be cured of HCV infection until 2030. Those with compensated advanced chronic liver disease remain at risk for hepatic decompensation and de novo HCC. Algorithms have been developed to stratify risk early after cure; however, data on long-term outcomes and the prognostic utility of these risk stratification algorithms at later time points are lacking. APPROACH AND RESULTS: We retrospectively analyzed a cohort of 2335 patients with compensated advanced chronic liver disease (liver stiffness measurement≥10 kPa) who achieved HCV-cure by interferon-free therapies from 15 European centers (median age 60.2±11.9 y, 21.1% obesity, 21.2% diabetes).During a median follow-up of 6 years, first hepatic decompensation occurred in 84 patients (3.6%, incidence rate: 0.74%/y, cumulative incidence at 6 y: 3.2%); 183 (7.8%) patients developed de novo HCC (incidence rate: 1.60%/y, cumulative incidence at 6 y: 8.3%), with both risks being strictly linear over time.Baveno VII criteria to exclude (FU-liver stiffness measurement <12 kPa and follow-up platelet count >150 g/L) or rule-in (FU-liver stiffness measurement ≥25 kPa) clinically significant portal hypertension (CSPH) stratified the risk of hepatic decompensation with proportional hazards. Estimated probability of CSPH discriminated patients developing versus not developing hepatic decompensation in the gray zone (ie, patients meeting none of the above criteria).Published HCC risk stratification algorithms identified high-incidence and low-incidence groups; however, the size of the latter group varied substantially (9.9%-69.1%). A granular "HCC-sustained virologic response" model was developed to inform an individual patient's HCC risk after HCV-cure. CONCLUSIONS: In patients with compensated advanced chronic liver disease, the risks of hepatic decompensation and HCC remain constant after HCV-cure, even in the long term (>3 y). One-time post-treatment risk stratification based on noninvasive criteria provides important prognostic information that is maintained during long-term follow-up, as the hazards remain proportional over time.

Characterizing Hepatitis Delta in Spain and the gaps in its management.

BACKGROUND AND AIMS: Chronic hepatitis D (CHD) is a severe form of chronic viral hepatitis. The estimated hepatitis delta prevalence in Spain is around 5% of patients with hepatitis B. Reimbursement of new antiviral therapies (Bulevirtide, BLV) was delayed in our country until February 2024. We aimed to characterize the clinical profile of patients with HDV/HBV infection in Spain and current barriers in their management at the time of BLV approval. METHOD: Multicenter registry including patients with positive anti-HDV serology actively monitored in 30 Spanish centers. Epidemiological, clinical and virological variables were recorded at the start of follow-up and at the last visit. RESULTS: We identified 329 anti-HDV patients, 41% were female with median age 51 years. The most common geographical origin was Spain (53%) and East Europe (24%). Patients from Spain were older and had HCV and HIV coinfection probably associated to past drug injection (p<0.01). HDV-RNA was positive in 138 of 221 assessed (62%). Liver cirrhosis was present at diagnosis in 33% and it was more frequent among viremic patients (58% vs 25%, p<0.01). After a median follow-up of 6 (3-12) years, 44 (16%) resolved infection (18 spontaneously and 26 after Peg-INF). An additional 10% of patients developed cirrhosis (n=137) during follow-up (45% had portal hypertension and 14% liver decompensation). Liver disease progression was associated to persisting viremia. CONCLUSION: One-third of the patients with CHD already have cirrhosis at diagnosis. Persistence of positive viremia is associated to rapid liver disease progression. Importantly, barriers to locally determine/quantify HDV-RNA were present.

Real-life effectiveness of sofosbuvir/velpatasvir/voxilaprevir in hepatitis C patients previously treated with sofosbuvir/velpatasvir or glecaprevir/pibrentasvir.

BACKGROUND: Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is the recommended rescue therapy for patients with chronic hepatitis C infection who fail direct-acting antivirals (DAAs). Data are limited on the effectiveness of this treatment after the current first-line therapies. Our aim was to analyse the effectiveness and safety of SOF/VEL/VOX among patients failing sofosbuvir/velpatasvir (SOF/VEL) or glecaprevir/pibrentasvir (GLE/PIB). METHODS: Retrospective multicentre study (26 Spanish hospitals), including chronic hepatitis C patients unsuccessfully treated with SOF/VEL or GLE/PIB, and retreated with SOF/VEL/VOX ± ribavirin for 12 weeks between December 2017 and December 2022. RESULTS: In total, 142 patients included: 100 (70.4%) had failed SOF/VEL and 42 (29.6%) GLE/PIB. Patients were mainly men (84.5%), White (93.9%), with hepatitis C virus genotype (GT) 3 (49.6%) and 47.2% had liver cirrhosis. Sustained virological response (SVR) was evaluated in 132 patients who completed SOF/VEL/VOX and were followed 12 weeks after end of treatment; 117 (88.6%) achieved SVR. There were no significant differences in SVR rates according to initial DAA treatment (SOF/VEL 87.9% vs. GLE/PIB 90.2%, p = 0.8), cirrhosis (no cirrhosis 90% vs. cirrhosis 87.1%, p = 0.6) or GT3 infection (non-GT3 91.9% vs. GT3 85.5%, p = 0.3). However, when considering the concurrent presence of SOF/VEL treatment, cirrhosis and GT3 infection, SVR rates dropped to 82.8%. Ribavirin was added in 8 (6%) patients, all achieved SVR. CONCLUSION: SOF/VEL/VOX is an effective rescue therapy for failures to SOF/VEL or GLE/PIB, with an SVR of 88.6%. Factors previously linked to lower SVR rates, such as GT3 infection, cirrhosis and first-line therapy with SOF/VEL were not associated with lower SVRs.

Rituximab is a safe and effective alternative treatment for patients with autoimmune hepatitis: Results from the ColHai registry.

BACKGROUND AND AIMS: Small series suggest that rituximab could be effective as treatment for autoimmune hepatitis (AIH), although data are scarce. We aimed to evaluate the efficacy and safety of rituximab in different cohorts of patients with AIH. METHODS: Multicentre retrospective analysis of the 35 patients with AIH and its variant forms treated with rituximab and included in the ColHai registry between 2015 and 2023. RESULTS: Most patients were female (83%), 10 (29%) had cirrhosis and four (11.4%) variant forms of AIH. Indication for rituximab were as follows: 14(40%) refractory AIH, 19(54%) concomitant autoimmune or haematological disorder, 2(6%) intolerance to prior treatments. In three (9%) subjects with a concomitant disorder, rituximab was the first therapy for AIH. Overall, 31 (89%) patients achieved or maintained complete biochemical response (CBR), including the three in first-line therapy. No difference in CBR was observed according to rituximab indication (refractory AIH 86% vs. concomitant disorders 90%, p = .824) or cirrhosis (80% vs. 92%, p = .319). Rituximab was associated with a significant reduction in corticosteroids (median dose: prior 20 vs. post 5 mg, p < .001) and the discontinuation of ≥1 immunosuppressant in 47% of patients. Flare-free rate at 1st, 2nd and 3rd year was 86%, 73% and 62% respectively. Flares were not associated with the development of liver failure and were successfully managed with repeated doses of rituximab and/or increased corticosteroids. Three (9%) patients experienced infusion-related adverse events (1 anaphylaxis and 2 flu-like symptoms) and five (14%) infections. CONCLUSION: Rituximab is safe and effective in patients with refractory AIH and those treated due to concomitant autoimmune or haematological disorders.

Post-treatment LSM rather than change during treatment predicts decompensation in patients with cACLD after HCV cure.

BACKGROUND & AIMS: Baveno VII has defined a clinically significant (i.e., prognostically meaningful) decrease in liver stiffness measurement (LSM) in cACLD as a decrease of ≥20% associated with a final LSM <20 kPa or any decrease to <10 kPa. However, these rules have not yet been validated against direct clinical endpoints. METHODS: We retrospectively analysed patients with cACLD (LSM ≥10 kPa) with paired liver stiffness measurement (LSM) before (BL) and after (FU) HCV cure by interferon-free therapies from 15 European centres. The cumulative incidence of hepatic decompensation was compared according to these criteria, considering hepatocellular carcinoma and non-liver-related death as competing risks. RESULTS: A total of 2,335 patients followed for a median of 6 years were analysed. Median BL-LSM was 16.6 kPa with 37.1% having ≥20 kPa. After HCV cure, FU-LSM decreased to a median of 10.9 kPa (<10 kPa: 1,002 [42.9%], ≥20 kPa: 465 [19.9%]) translating into a median LSM change of -5.3 (-8.8 to -2.4) kPa corresponding to -33.9 (-48.0 to -15.9) %. Patients achieving a clinically significant decrease (65.4%) had a significantly lower risk of hepatic decompensation (subdistribution hazard ratio: 0.12, 95% CI 0.04-0.35, p <0.001). However, these risk differences were primarily driven by a negligible risk in patients with FU-LSM <10 kPa (5-year cumulative incidence: 0.3%) compared to a high risk in patients with FU-LSM ≥20 kPa (16.6%). Patients with FU-LSM 10-19.9 kPa (37.4%) also had a low risk of hepatic decompensation (5-year cumulative incidence: 1.7%), and importantly, the risk of hepatic decompensation did not differ between those with/without an LSM decrease of ≥20% (p = 0.550). CONCLUSIONS: FU-LSM is key for risk stratification after HCV cure and should guide clinical decision making. LSM dynamics do not hold significant prognostic information in patients with FU-LSM 10-19.9 kPa, and thus, their consideration is not of sufficient incremental value in the specific context of HCV cure. IMPACT AND IMPLICATIONS: Liver stiffness measurement (LSM) is increasingly applied as a prognostic biomarker and commonly decreases in patients with compensated advanced chronic liver disease achieving HCV cure. Although Baveno VII proposed criteria for a clinically significant decrease, little is known about the prognostic utility of LSM dynamics (changes through antiviral therapy). Interestingly, in those with a post-treatment LSM of 10-19.9 kPa, LSM dynamics did not provide incremental information, arguing against the consideration of LSM dynamics as prognostic criteria. Thus, post-treatment LSM should guide the management of patients with compensated advanced chronic liver disease achieving HCV cure.

Outcomes and factors associated with relapse of vaccine-induced liver injury after SARS CoV-2 immunization: A nationwide study.

INTRODUCTION AND OBJECTIVES: Different patterns of liver injury have been reported in association with the SARS-CoV-2 vaccines. The aim of this study was to describe a nationwide cohort of patients with SARS CoV-2 vaccine-induced liver injury, focusing on treatment and the evolution after further booster administration. PATIENTS AND METHODS: multicentre, retrospective-prospective study, including subjects who developed abnormal liver tests within 90 days after administration of SARS-CoV-2 vaccination. RESULTS: 47 cases were collected: 17 after prime dose and 30 after booster. Age was 57 years, 30 (63.8 %) were female, and 7 (14.9 %) had a history of prior autoimmune hepatitis (AIH). Most cases were non-severe, though 9 (19.1 %) developed acute liver injury or failure (ALF). Liver injury tended to be more severe in those presenting after a booster (p=0.084). Pattern of liver injury was hepatocellular (80.9 %), mixed (12.8 %) and 3 (6.4 %) cholestatic. Liver biopsy was performed on 33 patients; 29 showed findings of AIH. Forty-one (87.2 %) patients received immunosuppressants, mostly corticosteroids (35/41). One required liver transplantation and another died due to ALF. Immunosuppression was discontinued in 6/41 patients without later rebound. Twenty-five subjects received at least one booster and 7 (28.0 %) relapsed from the liver injury, but all were non-severe. Recurrence was less frequent among patients on immunosuppressants at booster administration (28.6 % vs. 88.9 %, p=0.007). CONCLUSIONS: SARS CoV-2 vaccine-induced liver injury is heterogeneous but mostly immune-mediated. Relapse of liver injury after re-exposure to vaccine is frequent (28.0 %) but mild. Immunosuppression at booster administration is associated with a lower risk of liver injury.

Recent Advances and Potential Multi-Omics Approaches in the Early Phases of Inflammatory Bowel Disease.

Inflammatory bowel disease leads to debilitating gastrointestinal symptoms and reduced quality of life, resulting in a significant burden on healthcare utilization and costs. Despite substantial advancements in diagnosis and treatment, there may still be considerable delays in diagnosing some patients. To reduce disease progression before the full disease spectrum appears and improve prognostic outcomes, several strategies have concentrated on early intervention and prevention. Recent evidence shows that initial immune response changes and endoscopic lesions may exist for years before diagnosis, implying the existence of a preclinical phase of inflammatory bowel disease comparable to findings in other immune-mediated disorders. In this review, we highlight the most relevant findings regarding preclinical inflammatory bowel disease and the prospective role of novel omics techniques in this field.

Budesonide as first-line treatment in patients with autoimmune hepatitis seems inferior to standard predniso(lo)ne administration.

BACKGROUND AND AIMS: In patients with non-severe acute or chronic autoimmune hepatitis (AIH) without cirrhosis, clinical practice guidelines recommend indistinct use of prednisone or budesonide. However, budesonide is infrequently used in clinical practice. We aimed to describe its use and compare its efficacy and safety with prednisone as first-line options. APPROACH AND RESULTS: This was a retrospective, multicenter study of 105 naive AIH patients treated with budesonide as the first-line drug. The control group included 276 patients treated with prednisone. Efficacy was assessed using logistic regression and validated using inverse probability of treatment weighting propensity score. The median time to biochemical response (BR) was 3.1 months in patients treated with budesonide and 4.9 months in those with prednisone. The BR rate was significantly higher in patients treated with prednisone (87% vs. 49% of patients with budesonide, p < 0.001). The probability of achieving BR, assessed using the inverse probability of treatment weighting propensity score, was significantly lower in the budesonide group (OR = 0.20; 95% CI: 0.11-0.38) at any time during follow-up, and at 6 (OR = 0.51; 95% CI: 0.29-0.89) and 12 months after starting treatment (0.41; 95% CI: 0.23-0.73). In patients with transaminases <2 × upper limit of normal, BR was similar in both treatment groups. Prednisone treatment was significantly associated with a higher risk of adverse events (24.2% vs. 15.9%, p = 0.047). CONCLUSIONS: In the real-life setting, the use of budesonide as first-line treatment is low, and it is generally prescribed to patients with perceived less disease activity. Budesonide was inferior to prednisone as a first-line drug but was associated with fewer side effects.

Characterization of the perioperative changes of exosomal immune-related cytokines induced by prostatectomy in early-stage prostate cancer patients.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are relevant in prostate cancer microenvironment collaborating in tumor development. The main tumor marker used in this disease, prostate-specific antigen (PSA), does not provide information related to this tumor microenvironment. Cancer cells secrete exosomes carrying bioactive molecules contributing to MDSCs recruitment and induction. The aim of this study was to characterize the perioperative changes of exosomal cytokines relevant in MDSCs recruitment induced by prostatectomy in prostate cancer patients. METHODS: Blood was drawn from 26 early-stage prostate cancer patients before and after radical prostatectomy and from 16 healthy volunteers. Serum exosomes were separated by precipitation. Cytokines related with MDSC cell recruitment and activation CCL2, CXCL2, CXCL5, CXCL8, CXCL12, MIF, S100A9 and TGF-ß were measured in serum and serum-derived exosomes using immunometric assays. RESULTS: All cytokines were detected both in serum and exosomes, except for CXCL12, which was detected only in serum. Exosomes were enriched specially in MIF, TGF-ß and CXCL2. Presurgical MIF levels in exosomes correlated negatively with serum PSA. Also, presurgical TGF-ß decreased both in serum and exosomes as Gleason score rises. Patients presurgical exosomes had increased CCL2, CXCL5 and TGF-ß levels than exosomes from healthy controls. These differences were not observed when cytokines were analyzed in serum, except for TGF-ß. Cytokine levels of CCL2, CXCL5 decreased in patients' postsurgical exosomes, while TGF-ß further increased. On the contrary, S100A9 levels were lower in patients presurgical exosomes but increased after radical prostatectomy. CONCLUSIONS: Blood exosomal content in cytokines constitute an attractive source to evaluate MDSCs immunomodulators providing additional information related to tumor microenvironment in prostate cancer.

Blunted Reducing Power Generation in Erythrocytes Contributes to Oxidative Stress in Prepubertal Obese Children with Insulin Resistance.

Childhood obesity, and specifically its metabolic complications, are related to deficient antioxidant capacity and oxidative stress. Erythrocytes are constantly exposed to multiple sources of oxidative stress; hence, they are equipped with powerful antioxidant mechanisms requiring permanent reducing power generation and turnover. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are two key enzymes on the pentose phosphate pathway. Both enzymes supply reducing power by generating NADPH, which is essential for maintaining the redox balance within the cell and the activity of other antioxidant enzymes. We hypothesized that obese children with insulin resistance would exhibit blunted G6PDH and 6PGDH activities, contributing to their erythrocytes' redox status imbalances. We studied 15 control and 24 obese prepubertal children, 12 of whom were insulin-resistant according to an oral glucose tolerance test (OGTT). We analyzed erythroid malondialdehyde (MDA) and carbonyl group levels as oxidative stress markers. NADP+/NADPH and GSH/GSSG were measured to determine redox status, and NADPH production by both G6PDH and 6PGDH was assayed spectrophotometrically to characterize pentose phosphate pathway activity. Finally, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) activities were also assessed. As expected, MDA and carbonyl groups levels were higher at baseline and along the OGTT in insulin-resistant children. Both redox indicators showed an imbalance in favor of the oxidized forms along the OGTT in the insulin-resistant obese group. Additionally, the NADPH synthesis, as well as GR activity, were decreased. H2O2 removing enzyme activities were depleted at baseline in both obese groups, although after sugar intake only metabolically healthy obese participants were able to maintain their catalase activity. No change was detected in SOD activity between groups. Our results show that obese children with insulin resistance present higher levels of oxidative damage, blunted capacity to generate reducing power, and hampered function of key NADPH-dependent antioxidant enzymes.

Oxidative Stress in the Pathogenesis of Crohn's Disease and the Interconnection with Immunological Response, Microbiota, External Environmental Factors, and Epigenetics.

Inflammatory bowel disease (IBD) is a complex multifactorial disorder in which external and environmental factors have a large influence on its onset and development, especially in genetically susceptible individuals. Crohn's disease (CD), one of the two types of IBD, is characterized by transmural inflammation, which is most frequently located in the region of the terminal ileum. Oxidative stress, caused by an overabundance of reactive oxygen species, is present locally and systemically in patients with CD and appears to be associated with the well-described imbalanced immune response and dysbiosis in the disease. Oxidative stress could also underlie some of the environmental risk factors proposed for CD. Although the exact etiopathology of CD remains unknown, the key role of oxidative stress in the pathogenesis of CD is extensively recognized. Epigenetics can provide a link between environmental factors and genetics, and numerous epigenetic changes associated with certain environmental risk factors, microbiota, and inflammation are reported in CD. Further attention needs to be focused on whether these epigenetic changes also have a primary role in the pathogenesis of CD, along with oxidative stress.

Plasma Oncostatin M, TNF-α, IL-7, and IL-13 Network Predicts Crohn's Disease Response to Infliximab, as Assessed by Calprotectin Log Drop.

BACKGROUND: Cytokines emerge as possible biomarkers of response in Crohn's disease (CD). We aimed to determine the plasmatic cytokine profiles of active CD patients who started infliximab (IFX) treatment and their capacity to predict the response to IFX. METHODS: A total of 30 active CD patients receiving an induction therapy of IFX were enrolled in the study. Peripheral blood samples pretreatment were collected. Concentrations of 15 cytokines were measured by Luminex technology. Responses to IFX were evaluated by the drop in fecal calprotectin based on its logarithm-transformed values. A random forest (RF) predictive model was used for data analyses. RESULTS: Samples of 22 patients were analyzed. The RF model ranked the following cytokines as the top predictors of the response: tumor necrosis factor alpha (TNFα), interleukin (IL)-13, oncostatin M (OSM), and IL-7 (p < 0.005). Partial dependency plots showed that high levels of IL-13 pretreatment, low TNFα levels, and low IL-7 levels were associated with a favorable IFX response. Increased levels of OSM and TNFα predicted unfavorable responses to IFX. CONCLUSIONS: We here show that a log drop in calprotectin strongly correlates with clinical parameters and it can be proposed as a useful objective clinical response predictor. Plasma TNFα, IL-13, Il-7, and OSM network could predict CD response to IFX before induction therapy, as assessed by calprotectin log drop.

Performance comparison of two next-generation sequencing panels to detect actionable mutations in cell-free DNA in cancer patients.

Background Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Next-generation sequencing (NGS) technology provides the most complete mutational analysis, but nowadays limited data are available related to the comparison of results reported by different platforms. Here we compare two NGS panels for cfDNA: Oncomine™ Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360® (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory. Methods Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360® (G360) was requested as part of their clinical assistance. Blood samples were sent to be analyzed with G360 panel, and an additional blood sample was drawn to obtain and analyze cfDNA with Oncomine™ Pan-Cancer (OM) panel in an Ion GeneStudio S5™ System. Results cfDNA analysis globally rendered 101 mutations. Regarding the 55/101 mutations claimed to be included by manufacturers in both panels, 17 mutations were reported only by G360, 10 only by OM and 28 by both. In those coincident cases, there was a high correlation between the variant allele fractions (VAFs) calculated with each panel (r = 0.979, p < 0.01). Regarding the six actionable mutations with an FDA-approved therapy reported by G360, one was missed with OM. Also, 12 mutations with clinical trials available were reported by G360 but not by OM. Conclusions In summary, G360 and OM can produce different mutational profile in the same sample, even in genes included in both panels, which is especially important if these mutations are potentially druggable.

Genomewide Association Study of Severe Covid-19 with Respiratory Failure.

BACKGROUND: There is considerable variation in disease behavior among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (Covid-19). Genomewide association analysis may allow for the identification of potential genetic factors involved in the development of Covid-19. METHODS: We conducted a genomewide association study involving 1980 patients with Covid-19 and severe disease (defined as respiratory failure) at seven hospitals in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe. After quality control and the exclusion of population outliers, 835 patients and 1255 control participants from Italy and 775 patients and 950 control participants from Spain were included in the final analysis. In total, we analyzed 8,582,968 single-nucleotide polymorphisms and conducted a meta-analysis of the two case-control panels. RESULTS: We detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2, which were significant at the genomewide level (P<5×10-8) in the meta-analysis of the two case-control panels (odds ratio, 1.77; 95% confidence interval [CI], 1.48 to 2.11; P = 1.15×10-10; and odds ratio, 1.32; 95% CI, 1.20 to 1.47; P = 4.95×10-8, respectively). At locus 3p21.31, the association signal spanned the genes SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1. The association signal at locus 9q34.2 coincided with the ABO blood group locus; in this cohort, a blood-group-specific analysis showed a higher risk in blood group A than in other blood groups (odds ratio, 1.45; 95% CI, 1.20 to 1.75; P = 1.48×10-4) and a protective effect in blood group O as compared with other blood groups (odds ratio, 0.65; 95% CI, 0.53 to 0.79; P = 1.06×10-5). CONCLUSIONS: We identified a 3p21.31 gene cluster as a genetic susceptibility locus in patients with Covid-19 with respiratory failure and confirmed a potential involvement of the ABO blood-group system. (Funded by Stein Erik Hagen and others.).

Bases for the Adequate Development of Nutritional Recommendations for Patients with Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory condition of the gastrointestinal tract; it is a heterogeneous and multifactorial disorder resulting from a complex interplay between genetic variation, intestinal microbiota, the host immune system and environmental factors such as diet, drugs, breastfeeding and smoking. The interactions between dietary nutrients and intestinal immunity are complex. There is a compelling argument for environmental factors such as diet playing a role in the cause and course of IBD, given that three important factors in the pathogenesis of IBD can be modulated and controlled by diet: intestinal microbiota, the immune system and epithelial barrier function. The aim of this review is to summarize the epidemiological findings regarding diet and to focus on the effects that nutrients exert on the intestinal mucosa-microbiota-permeability interaction. The nature of these interactions in IBD is influenced by alterations in the nutritional metabolism of the gut microbiota and host cells that can influence the outcome of nutritional intervention. A better understanding of diet-host-microbiota interactions is essential for unravelling the complex molecular basis of epigenetic, genetic and environmental interactions underlying IBD pathogenesis as well as for offering new therapeutic approaches for the treatment of IBD.

Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis.

Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.

The Dynamic Use of EGFR Mutation Analysis in Cell-Free DNA as a Follow-Up Biomarker during Different Treatment Lines in Non-Small-Cell Lung Cancer Patients.

Epidermal growth factor receptor (EGFR) mutational testing in advanced non-small-cell lung cancer (NSCLC) is usually performed in tumor tissue, although cfDNA (cell-free DNA) could be an alternative. We evaluated EGFR mutations in cfDNA as a complementary tool in patients, who had already known EGFR mutations in tumor tissue and were treated with either EGFR-tyrosine kinase inhibitors (TKIs) or chemotherapy. We obtained plasma samples from 21 advanced NSCLC patients with known EGFR tumor mutations, before and during therapy with EGFR-TKIs and/or chemotherapy. cfDNA was isolated and EGFR mutations were analyzed with the multiple targeted cobas EGFR Mutation Test v2. EGFR mutations were detected at baseline in cfDNA from 57% of patients. The semiquantitative index (SQI) significantly decreased from the baseline (median = 11, IQR = 9.5-13) to the best response (median = 0, IQR = 0-0, p < 0.01), followed by a significant increase at progression (median = 11, IQR = 11-15, p < 0.01) in patients treated with either EGFR-TKIs or chemotherapy. The SQI obtained with the cobas EGFR Mutation Test v2 did not correlate with the concentration in copies/mL determined by droplet digital PCR. Resistance mutation p.T790M was observed at progression in patients with either type of treatment. In conclusion, cfDNA multiple targeted EGFR mutation analysis is useful for treatment monitoring in tissue of EGFR-positive NSCLC patients independently of the drug received.

Group-Level Progressive Alterations in Brain Connectivity Patterns Revealed by Diffusion-Tensor Brain Networks across Severity Stages in Alzheimer's Disease.

Alzheimer's disease (AD) is a chronically progressive neurodegenerative disease highly correlated to aging. Whether AD originates by targeting a localized brain area and propagates to the rest of the brain across disease-severity progression is a question with an unknown answer. Here, we aim to provide an answer to this question at the group-level by looking at differences in diffusion-tensor brain networks. In particular, making use of data from Alzheimer's Disease Neuroimaging Initiative (ADNI), four different groups were defined (all of them matched by age, sex and education level): G1 (N1 = 36, healthy control subjects, Control), G2 (N2 = 36, early mild cognitive impairment, EMCI), G3 (N3 = 36, late mild cognitive impairment, LMCI) and G4 (N4 = 36, AD). Diffusion-tensor brain networks were compared across three disease stages: stage I (Control vs. EMCI), stage II (Control vs. LMCI) and stage III (Control vs. AD). The group comparison was performed using the multivariate distance matrix regression analysis, a technique that was born in genomics and was recently proposed to handle brain functional networks, but here applied to diffusion-tensor data. The results were threefold: First, no significant differences were found in stage I. Second, significant differences were found in stage II in the connectivity pattern of a subnetwork strongly associated to memory function (including part of the hippocampus, amygdala, entorhinal cortex, fusiform gyrus, inferior and middle temporal gyrus, parahippocampal gyrus and temporal pole). Third, a widespread disconnection across the entire AD brain was found in stage III, affecting more strongly the same memory subnetwork appearing in stage II, plus the other new subnetworks, including the default mode network, medial visual network, frontoparietal regions and striatum. Our results are consistent with a scenario where progressive alterations of connectivity arise as the disease severity increases and provide the brain areas possibly involved in such a degenerative process. Further studies applying the same strategy to longitudinal data are needed to fully confirm this scenario.

Lagged and instantaneous dynamical influences related to brain structural connectivity.

Contemporary neuroimaging methods can shed light on the basis of human neural and cognitive specializations, with important implications for neuroscience and medicine. Indeed, different MRI acquisitions provide different brain networks at the macroscale; whilst diffusion-weighted MRI (dMRI) provides a structural connectivity (SC) coincident with the bundles of parallel fibers between brain areas, functional MRI (fMRI) accounts for the variations in the blood-oxygenation-level-dependent T2(*) signal, providing functional connectivity (FC). Understanding the precise relation between FC and SC, that is, between brain dynamics and structure, is still a challenge for neuroscience. To investigate this problem, we acquired data at rest and built the corresponding SC (with matrix elements corresponding to the fiber number between brain areas) to be compared with FC connectivity matrices obtained by three different methods: directed dependencies by an exploratory version of structural equation modeling (eSEM), linear correlations (C) and partial correlations (PC). We also considered the possibility of using lagged correlations in time series; in particular, we compared a lagged version of eSEM and Granger causality (GC). Our results were two-fold: firstly, eSEM performance in correlating with SC was comparable to those obtained from C and PC, but eSEM (not C, nor PC) provides information about directionality of the functional interactions. Second, interactions on a time scale much smaller than the sampling time, captured by instantaneous connectivity methods, are much more related to SC than slow directed influences captured by the lagged analysis. Indeed the performance in correlating with SC was much worse for GC and for the lagged version of eSEM. We expect these results to supply further insights to the interplay between SC and functional patterns, an important issue in the study of brain physiology and function.