In a previous clinical study, a probiotic formulation (PF) consisting ofLactobacillus helveticusR0052 andBifidobacterium longumR0175 (PF) decreased stress-induced gastrointestinal discomfort. Emerging evidence of a role for gut microbiota on central nervous system functions therefore suggests that oral intake of probiotics may have beneficial consequences on mood and psychological distress. The aim of the present study was to investigate the anxiolytic-like activity of PF in rats, and its possible effects on anxiety, depression, stress and coping strategies in healthy human volunteers. In the preclinical study, rats were daily administered PF for 2 weeks and subsequently tested in the conditioned defensive burying test, a screening model for anti-anxiety agents. In the clinical trial, volunteers participated in a double-blind, placebo-controlled, randomised parallel group study with PF administered for 30 d and assessed with the Hopkins Symptom Checklist (HSCL-90), the Hospital Anxiety and Depression Scale (HADS), the Perceived Stress Scale, the Coping Checklist (CCL) and 24 h urinary free cortisol (UFC). Daily subchronic administration of PF significantly reduced anxiety-like behaviour in rats (P < 0·05) and alleviated psychological distress in volunteers, as measured particularly by the HSCL-90 scale (global severity index,P < 0·05; somatisation,P < 0·05; depression,P < 0·05; and anger–hostility,P < 0·05), the HADS (HADS global score,P < 0·05; and HADS-anxiety,P < 0·06), and by the CCL (problem solving,P < 0·05) and the UFC level (P < 0·05).L. helveticusR0052 andB. longumR0175 taken in combination display anxiolytic-like activity in rats and beneficial psychological effects in healthy human volunteers.

Aims/hypothesis: Coronavirus disease-2019 (COVID-19) is a life-threatening infection caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Diabetes has rapidly emerged as a major comorbidity for COVID-19 severity. However, the phenotypic characteristics of diabetes in COVID-19 patients are unknown. Methods: We conducted a nationwide multicentre observational study in people with diabetes hospitalised for COVID-19 in 53 French centres in the period 10–31 March 2020. The primary outcome combined tracheal intubation for mechanical ventilation and/or death within 7 days of admission. Age- and sex-adjusted multivariable logistic regressions were performed to assess the prognostic value of clinical and biological features with the endpoint. ORs are reported for a 1 SD increase after standardisation. Results: The current analysis focused on 1317 participants: 64.9% men, mean age 69.8 ± 13.0 years, median BMI 28.4 (25th–75th percentile: 25.0–32.7) kg/m²; with a predominance of type 2 diabetes (88.5%). Microvascular and macrovascular diabetic complications were found in 46.8% and 40.8% of cases, respectively. The primary outcome was encountered in 29.0% (95% CI 26.6, 31.5) of participants, while 10.6% (9.0, 12.4) died and 18.0% (16.0, 20.2) were discharged on day 7. In univariate analysis, characteristics prior to admission significantly associated with the primary outcome were sex, BMI and previous treatment with renin–angiotensin–aldosterone system (RAAS) blockers, but not age, type of diabetes, HbA_1c, diabetic complications or glucose-lowering therapies. In multivariable analyses with covariates prior to admission, only BMI remained positively associated with the primary outcome (OR 1.28 [1.10, 1.47]). On admission, dyspnoea (OR 2.10 [1.31, 3.35]), as well as lymphocyte count (OR 0.67 [0.50, 0.88]), C-reactive protein (OR 1.93 [1.43, 2.59]) and AST (OR 2.23 [1.70, 2.93]) levels were independent predictors of the primary outcome. Finally, age (OR 2.48 [1.74, 3.53]), treated obstructive sleep apnoea (OR 2.80 [1.46, 5.38]), and microvascular (OR 2.14 [1.16, 3.94]) and macrovascular complications (OR 2.54 [1.44, 4.50]) were independently associated with the risk of death on day 7. Conclusions/interpretations: In people with diabetes hospitalised for COVID-19, BMI, but not long-term glucose control, was positively and independently associated with tracheal intubation and/or death within 7 days. Trial registration: clinicaltrials.gov NCT04324736.

Aims/hypothesis: This is an update of the results from the previous report of the CORONADO (Coronavirus SARS-CoV-2 and Diabetes Outcomes) study, which aims to describe the outcomes and prognostic factors in patients with diabetes hospitalised for coronavirus disease-2019 (COVID-19). Methods: The CORONADO initiative is a French nationwide multicentre study of patients with diabetes hospitalised for COVID-19 with a 28-day follow-up. The patients were screened after hospital admission from 10 March to 10 April 2020. We mainly focused on hospital discharge and death within 28 days. Results: We included 2796 participants: 63.7% men, mean age 69.7 ± 13.2 years, median BMI (25th–75th percentile) 28.4 (25.0–32.4) kg/m². Microvascular and macrovascular diabetic complications were found in 44.2% and 38.6% of participants, respectively. Within 28 days, 1404 (50.2%; 95% CI 48.3%, 52.1%) were discharged from hospital with a median duration of hospital stay of 9 (5–14) days, while 577 participants died (20.6%; 95% CI 19.2%, 22.2%). In multivariable models, younger age, routine metformin therapy and longer symptom duration on admission were positively associated with discharge. History of microvascular complications, anticoagulant routine therapy, dyspnoea on admission, and higher aspartate aminotransferase, white cell count and C-reactive protein levels were associated with a reduced chance of discharge. Factors associated with death within 28 days mirrored those associated with discharge, and also included routine treatment by insulin and statin as deleterious factors. Conclusions/interpretation: In patients with diabetes hospitalised for COVID-19, we established prognostic factors for hospital discharge and death that could help clinicians in this pandemic period. Trial registration: Clinicaltrials.gov identifier: NCT04324736 Graphical

Background—: Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. Methods and Results—: We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% ( P <0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients ( P =0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline ( P <0.0001). Conclusions—: PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. Clinical Trial Registration—: URL: http://www.clinicaltrials.gov . Unique Identifier: NCT01604824.

Objective—Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)–neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. Approach and Results—Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial. Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. Conclusions—Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (−38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (−89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19±0.77-fold in HLC-S127R compared to 1.38±0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.