Neutralizing IFN-γ autoantibodies are rare and pathogenic in HLA-DRB1*15:02 or 16:02 individuals.

BACKGROUNDWeakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adults harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGAs in the general population is unknown, as are the penetrance of nAIGAs in HLA-DRB1*15:02 or 16:02 individuals and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs.METHODSThis study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM.RESULTSWe found that anti-IFN-γ auto-Abs detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype were not neutralizing. Moreover, we did not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02. Additionally, nAIGAs were absent in 1,063 patients with an autoimmune disease. Finally, 7 of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGAs.CONCLUSIONThese findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are the cause of EM disease.FUNDINGThe Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983 and U19AIN1625568), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), the French National Research Agency (ANR) under the "Investments for the Future" program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), ANR-GENMSMD (ANR-16-CE17-0005-01), ANR-MAFMACRO (ANR-22-CE92-0008), ANRSECTZ170784, the French Foundation for Medical Research (FRM) (EQU201903007798), the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AI2D (ANR-22-CE15-0046) projects, the ANR-RHU program (ANR-21-RHUS-08-COVIFERON), the European Union's Horizon 2020 research and innovation program under grant agreement no. 824110 (EASI-genomics), the Square Foundation, Grandir - Fonds de solidarité pour l'enfance, the Fondation du Souffle, the SCOR Corporate Foundation for Science, the Battersea & Bowery Advisory Group, William E. Ford, General Atlantic's Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic's Co-President, Managing Director, and Head of business in EMEA, and the General Atlantic Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM) and of Paris Cité University. JR was supported by the INSERM PhD program for doctors of pharmacy (poste d'accueil INSERM). JR and TLV were supported by the Bettencourt-Schueller Foundation and the MD-PhD program of the Imagine Institute. MO was supported by the David Rockefeller Graduate Program, the Funai Foundation for Information Technology (FFIT), the Honjo International Scholarship Foundation (HISF), and the New York Hideyo Noguchi Memorial Society (HNMS).

Plasma Retinol Concentration Is Mainly Driven by Transthyretin in Hemodialysis Patients.

OBJECTIVE: Micronutrients deficiencies in hemodialysis patients are due to low dietary intakes and intradialytic losses for hydrophilic micronutrients. Conversely, lipophilic nondialyzable compounds might accumulate because of a lack of elimination through renal metabolism or dialysis. Other compounds have complex metabolism: their concentration is not explained by these phenomenons. The aim of this study was to report plasma concentrations of lipophilic micronutrients in hemodialysis patients and to analyze if these concentrations were predictive of mortality. DESIGN: The design was monocentric observational longitudinal study. SUBJECTS: A total of 123 hemodialysis patients included in this observational study. MAIN OUTCOME MEASURE: Plasma concentration of lipophilic micronutrients retinol and its two co-transporters transthyretin and retinol-binding protein 4, tocopherol, and carotenoids (α-carotene and ß-carotene, ß-cryptoxanthin, lycopene, lutein, and zeaxanthin), and all factors associated with 1-year mortality. RESULTS: Within the 123 patients of the study, median age (interquartile range) was 77.5 (69.5-84.5) years and 58.5% were male. Median retinol plasma concentration was 4.07 (2.65-5.51) µmol/L, and 91.9% of patient had high plasma retinol concentrations. In monovariate analysis, retinol levels were inversely correlated with mortality (hazard ratio = 0.57 [0.45-0.72]; P < .001). This effect remained significant after adjustment with several parameters. Nevertheless, the correlation between retinol and mortality disappeared as soon as transthyretin was added in the statistical model, suggesting an effect of transthyretin as confusing bias. Median tocopherol plasma concentration was 34.8 (28.3-42.9) µmol/L and 72.4% of patients had high plasma tocopherol concentration. Neither tocopherol plasma levels nor carotenoids concentrations were correlated with death in multivariate analysis. CONCLUSIONS: In hemodialysis patients, the correlation between retinol plasma concentration and mortality represents the nutritional status but not a direct biological effect of retinol. Retinol is only a surrogate predictor of mortality. It might not represent vitamin A levels, but likely the transthyretin level. Plasma retinol levels should be interpreted cautiously in hemodialysis patients.

Haemodialysis patients with diabetes eat less than those without: A plea for a permissive diet.

AIM: The main cause of malnutrition in haemodialysis patients is a spontaneous decline in energy and protein intakes. This study aims to report the dietary energy intake (DEI), dietary protein intake (DPI), and dietary micronutrient intake in a French HD population, to report factors associated with a low DPI and DEI, and to analyze if nutritional intake was correlated with nutritional status. METHODS: We conducted an observational cross-sectional study in a haemodialysis population of 87 adult patients in July 2014. Daily nutritional oral intake, handgrip strength, body composition measured by bioimpedancemetry, and biological and dialysis parameters were obtained from medical records. Statistical analyses of parameters associated with DEI and DPI were performed. RESULTS: The median age (interquartile range) of the population was 77.3 [71.1; 84.8] years, 57.5% were men, and 52.9% had diabetes mellitus. Median weight-adjusted DEI was 18.4 [15.7;22.3] kcal/kg per day (1308 [1078; 1569] kcal/day), and median weight-adjusted DPI was 0.80 [0.66; 0.96] g/kg per day (57.5 [47.1; 66.8] g/day). In multivariate analysis, weight-adjusted DEI was statistically lower in patients with diabetes (coefficient [95%CI] -3.81[-5.21;-2.41] kcal/kg per day; P = 0.01) but was not associated with the others parameters. When DEI was not adjusted for weight, diabetes was no longer associated with DEI, but female gender (-178[-259;-961] kcal/day; P = 0.03) and a higher Charlson comorbidity index (-30[-44;-15]; P = 0.04) were associated with a lower calorie intake. Results for DPI were similar except that the Charlson comorbidity index did not reach significance. CONCLUSIONS: Diabetes is an important factor associated with low dietary intake in haemodialysis patients. Restrictive regimens should be prescribed cautiously in haemodialysis patients, especially in those with diabetes.

The influence of the number of cells scored on the sensitivity in the comet assay.

The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different chemicals were used: Caco-2 cells were exposed to ethylmethane sulfonate and hydrogen peroxide in three concentrations, and HepG2 cells were exposed to ethylmethane sulfonate, hydrogen peroxide and benzo[a]pyrene in up to four concentrations, in four to five independent experiments. The scoring was carried out by means of a fully automated scoring system and the results were analyzed by evaluating the % tail DNA of 100-700 randomly selected cells for each slide consisting of two gels. By increasing the number of cells scored, the coefficients of variance decreased, leading to an improved sensitivity of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables "the number of cells scored" and "concentration" on the total variation in the coefficients of variance dataset was statistically significant (p<0.05). The increase in sensitivity was demonstrated by the possibility to detect an increase in % tail DNA with statistical significance at lower concentrations. The results indicated that for low levels of DNA damage, below 9% tail DNA, scoring of 600 cells increased the sensitivity compared with scoring of 100 cells. For relatively low levels of DNA damage, about 9-16% tail DNA, scoring of 300 cells increased the sensitivity. Thus, the recommendation for the optimum number of cells scored would be 600 and 300 for low and relatively low levels of DNA damage, respectively. The findings from this study could be particularly important for bio-monitoring studies where small differences in DNA-damage levels could be relevant.

The influence of scoring method on variability in results obtained with the comet assay.

As part of a project to develop high throughput versions of the comet assay (single cell gel electrophoresis), with a consequent need for more efficient scoring, we have compared the performance of visual scoring, automated and semi-automated image analysis when assessing comets in the same set of gels from dose-response experiments with typical DNA-damaging agents. Human lymphoblastoid TK-6 cells were treated with concentrations of methylmethanesulphonate between 0.04 and 0.6 mM, and peripheral human lymphocytes were incubated, after embedding in agarose, with H(2)O(2) concentrations from 2.5 to 160 µM. All three scoring methods proved capable of detecting a significant level of damage at the lowest concentration of each agent. Visual scoring systematically overestimates low levels of damage compared with computerised image analysis; on the other hand, heavily damaged comets are less efficiently detected with image analysis. Overall, the degree of agreement between the scoring methods is within acceptable limits according to a Bland-Altman analysis.

Nicotine enhances intracellular nicotinic receptor maturation: a novel mechanism of neural plasticity?

Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain alpha4beta2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This "maturational enhancer" action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions.

Nicotine upregulates its own receptors through enhanced intracellular maturation.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker's brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine.

In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.