Protein-S-100-beta is increased in patients with decompensated cirrhosis admitted to ICU.

Background: Hepatic encephalopathy (HE) is highly prevalent in patients with liver diseases. The pathophysiology of HE is centered on the synergic role of hyperammonemia and systemic inflammation. However, some data suggest altered functioning of the blood-brain barrier (BBB). Assessing BBB function is challenging in clinical practice and at the bedside. Protein-S-100 Beta (PS100-Beta) could be a useful peripheral marker of BBB permeability in HE. This study aimed to assess plasmatic PS100-Beta levels in a prospective cohort of patients admitted to the intensive care unit (ICU) with decompensated cirrhosis with and without overt HE. Methods: We retrospectively evaluated a prospective cohort of cirrhotic patients admitted to the ICU from October 2013 to September 2015 that had an available plasmatic PS100-Beta measurement. Patients with previous neurological impairment or limitation of intensive or resuscitative measures were excluded. Overt HE was defined as West-Haven grades 2 to 4. The patients were compared to a control cohort of outpatient clinic cirrhotic and non-cirrhotic patients explored for isolated elevation of liver enzymes. After ICU discharge, the patients were followed for at least 3 months for the occurrence of overt HE. Adverse outcomes (liver transplantation or death) were collected. The ability of PS100-Beta - in combination with other factors - to predict overt HE was evaluated in a multivariate analysis using logistic regression. Likelihood ratios were used to determine the effects and calculate odds ratios (OR). Survival analysis was performed by using the Kaplan-Meier method and survival between groups was compared using a Log-rank test. Results: A total of 194 ICU patients and 207 outpatients were included in the study. Increased levels of plasmatic PS100-Beta were detected in the ICU decompensated cirrhotic patients compared with the outpatients ([0.15±0.01] mg/L vs. [0.08±0] mg/L, P <0.001). ICU patients with overt HE had higher levels of PS100-Beta ([0.19±0.03] mg/L) compared with the ICU patients without overt HE ([0.13±0.01] mg/L) (P=0.003). PS100-Beta levels did not differ in outpatients with F 0-3 compared to F 4 fibrosis (P=0.670). PS100-Beta values were correlated with Child-Pugh score (P <0.001), Model for End-Stage Liver Disease (MELD) score (P=0.004), C-reactive protein (P <0.001), ammonemia (P <0.001), and chronic liver failure consortium (CLIF-C) organ failure (P <0.001) and CLIF-C acute-on-chronic (P=0.038) scores, but not with leukocytes (P=0.053), procalcitonin (PCT) (P=0.107), or the lymphocyte-to-neutrophil ratio in ICU patients (P=0.522). In a multivariate model including age, ammonemia, PS100-Beta, PCT, MELD, presence of transjugular portosystemic shunt, and sodium level, the diagnostic performance was 0.765 for the diagnosis of overt HE. Patients with a PS100-Beta level <0.12 mg/L had a better overall survival (P=0.019) and a better survival without liver transplantation (P=0.013). Conclusions: Serum levels of PS100-Beta are elevated in ICU patients with decompensated cirrhosis, and even more so in those displaying overt HE, and the levels are correlated with outcome. This suggests an increase in the permeability of the BBB in these patients.

Burden of grade 3 or 4 liver injury associated with immune checkpoint inhibitors.

Background & Aims: There is concern about the burden of liver injury in patients with cancer exposed to immune checkpoints inhibitors (ICIs). Methods: In a retrospective cohort study, we evaluated the likelihood of grade 3/4 liver injury, of grade 3/4 cholestatic liver injury, and of liver failure, as per the Common Terminology Criteria for Adverse Events (CTCAE) version 5, following treatment with ICIs. We compared these occurrences with a group of cancer patients who were propensity-matched and treated with conventional chemotherapy. For all ICI patients experiencing grade 3/4 liver injury, we conducted a causality assessment using the RUCAM method and examined patient outcomes. Results: Among 952 patients (median [IQR] age 66 [57-73] years, 64% males) who were treated with ICI between January 1, 2015, and December 31, 2019, a total of 86 (9%) progressed to grade 3/4 liver injury, and liver failure was not observed. Anti-PD-(L)1/anti-CTLA-4 antibodies combinations (adjusted hazard ratio 3.36 [95% CI: 1.67-6.79]; p <0.001), and chronic hepatitis B (adjusted hazard ratio 5.48 [95% CI: 1.62-18.5]; p = 0.006], were independent risk factors. Liver injury was attributed to ICI treatment in 19 (2.0%) patients. Patients with ICI toxicity typically presented with granulomatous hepatitis or cholangiocyte inflammation. ICI withdrawal was associated with cancer progression and mortality. Re-introduction of ICI was not associated with recurrent grade 3/4 liver injury. Compared with matched patients treated with conventional, non-ICI-based chemotherapy, anti-PD-(L)1/anti-CTLA-4 combinations (p <0.001) and anti-PD-(L)1 monotherapies (p = 0.053) increased the risk of grade 3/4 liver injury and of grade 3/4 cholestatic liver injury, respectively. Conclusions: An increased risk of grade 3/4 liver injury under anti-PD-(L)1/anti-CTLA-4 antibodies was observed, whereas no substantial increase in the likelihood of liver failure occurred even after treatment reintroduction. Impact and implications: There is concern about liver injury in patients with cancer exposed to immune checkpoints inhibitors (ICIs). We investigated the burden of grade 3/4 liver injury after treatment with ICIs in a multicentric cohort of patients with cancer. Overall, a 9% incidence of grade 3/4 liver injury was detected after ICIs, and direct ICI hepatotoxicity was demonstrated in 2% of patients. Anti-PD-(L)1/Anti-CTLA-4 antibody combinations, and chronic HBV infection were independent risk factors. ICI withdrawal for grade 3/4 liver injury was associated with cancer progression. Re-introduction of ICI treatment was not associated with recurrent grade 3/4 liver injury.

Clinical and genetic definition of serum bilirubin levels for the diagnosis of Gilbert syndrome and hypobilirubinemia.

BACKGROUND AND AIMS: Gilbert syndrome (GS) is genotypically predetermined by UGT1A1*28 homozygosity in Europeans and is phenotypically defined by hyperbilirubinemia using total bilirubin (TB) cutoff ≥1mg/dL (17 µmol/L). The prevalence of illnesses associated with GS and hypobilirubinemia has never been studied prospectively. As TB varies with UGT1A1*28 genotyping, sex, and age, we propose stratified definitions of TB reference intervals and report the prevalence of illnesses and adjusted 15 years survival. METHODS: UK Biobank with apparently healthy liver participants (middle-aged, n=138,125) were analyzed after the exclusion of of nonhealthy individuals. The stratified TB was classified as GS when TB >90th centile; <10th centile indicated hypobilirubinemia, and between the 10th and 90th centile was normobilirubinemia. We compared the prevalence and survival rates of 54 illnesses using odds ratio (OR), logistic regression, and Cox models adjusted for confounders, and causality by Mendelian randomizations. RESULTS: In women, we identified 10% (7,741/76,809) of GS versus 3.7% (2,819/76,809) using the historical cutoff of ≥1 mg/dL (P<0.0001). When GS and hypobilirubinemia participants were compared with normobilirubinemia, after adjustment and Mendelian randomizations, only cholelithiasis prevalence was significantly higher (OR=1.50; 95% CI [1.3-1.7], P=0.001) in men with GS compared with normobilirubinemia and in causal association with bilirubin (P=0.04). No adjusted survival was significantly associated with GS or hypobilirubinemia. CONCLUSIONS: In middle-aged Europeans, the stratified TB demonstrates a careless GS underestimation in women when using the standard unisex 1 mg/dL cutoff. The prevalence of illnesses is different in GS and hypobilirubinemia as well as survivals before adjusting for confounding factors. With the exception of cholelithiasis in men, these differences were no more significant after adjustment and Mendelian randomization.

[Impact of voxelotor on hemoglobin electrophoretic and chromatographic profiles]. / Conséquences du traitement par voxelotor sur les profils électrophorétiques et chromatographiques de l'hémoglobine.

Voxelotor (GBT440, OXBRYTA®) appeared recently as one of the possible treatments for sickle cell disease. This molecule, by binding the alpha globin of hemoglobin, causes hyperaffinity of the latter for oxygen and reduces its polymerization properties. Several therapeutic trials have been able to show its effectiveness on certain aspects of sickle cell disease; thus, the french HAS (High Authority of Health) college issued an early access authorization and, since 2021, this treatment can be offered to patients under a temporary authorization for use. Consequently, the laboratories that carry out the biological monitoring of sickle cell patients will be confronted with new profiles characteristic of the presence of hemoglobin combined with GBT440. This work presents a collection of images obtained by different techniques: HPLC, capillary electrophoresis, isoelectrofocusing, alkaline gel and acid agar gel electrophoresis in transfused or non-transfused sickle cell disease patients. The ability to observe the presence of GBT440 by these analyzes could be useful in order to characterize the therapeutic follow-up of patients.

Diagnosis of chylous abdominal effusions: what is the triglyceride threshold value?

Introduction: Chylous abdominal effusions are serious complications that can be triggered by various aetiologies. The biochemical diagnosis of chyle leakage in ascites or in peritoneal fluid capsules relies on the detection of chylomicrons. Assaying the fluid's concentration of triglycerides is still the first-line tool. Given that only one comparative study has sought to quantify the value of the triglyceride assay for diagnosing chylous ascites in humans, our objective was to provide practical triglyceride thresholds. Materials and methods: We conducted a 9-year, retrospective, single-centre study of adult patients and compared a triglyceride assay with lipoprotein gel electrophoresis for the analysis of 90 non-recurring abdominal effusions (ascites and abdominal collections) of which 65 were chylous. Results: A triglyceride threshold of 0.4 mmol/L was associated with a sensitivity > 95%, and a threshold of 2.4 mmol/L was associated with a specificity > 95%. According to Youden index, the best threshold was 0.65 mmol/L with a sensitivity of 88 (77-95)%, a specificity of 72 (51-88)%, and, in our series, a positive predictive value of 89 (79-95)% and a negative predictive value of 69 (48-86)%. Conclusions: In our series, cut-off of 0.4 mmol/L could be used for ruling-out diagnosis of chylous effusions, while cut-off of 2.4 mmol/L could be used for reasonably confirming diagnosis.

Could the chylomicron marker apoB48 be of value in the diagnosis of chylous effusions?

BACKGROUND: Chylous effusions such as chylothorax, chylopericardium and chylous ascites are marked by the abnormal presence of chylomicrons in serous membranes. These relatively rare situations are associated with high morbidity and mortality rates. Given that a macroscopic assessment of the fluid is insufficient, the current gold standard method for chylous effusion is the electrophoretic separation of lipoproteins. Serous effusions are most frequently assayed for triglycerides, with a diagnostic threshold varying between studies. The present study is the first to assess the value of the apolipoprotein B48, specific of the chylomicron, in the diagnosis of chylous effusions. METHODS: A chemiluminescent sandwich enzyme immunoassay was used to measure levels of apoB48 in remnant samples of effusion fluid sent to our laboratory for chylomicron detection and lipid assays. The diagnostic values of apoB48 and triglyceride assays were compared with that of the gold standard method. RESULTS: The triglyceride and apoB48 levels and the triglyceride/cholesterol ratio in the effusion fluid were significantly higher in patients with chylous effusion. The threshold values for apoB48 were respectively 2.45, 0.25 and 19.00 µg/mL for a maximal Youden index, a sensitivity > 95 %, and a specificity > 95 %. The apoB48 assay's diagnostic value might be at least as high as that of a triglyceride assay (area under the receiver operating characteristic curve [95 % confidence interval]: 0.84 [0.72, 0.96]) and 0.80 [0.67, 0.94], respectively). CONCLUSION: ApoB48 appears to be a promising marker for the diagnosis of chylous effusions; the putative diagnostic improvement must be confirmed in larger studies.

Chylopericardium or contamination by injectable lipid emulsion? / « Mets la main sur mon cœur, Et vois comme il se trouble ¼ ­ un pseudo-chylopéricarde ?

Effusions can show some surprises. We document the case of a fourteen-month-old male patient with short-bowel syndrome, hospitalized in a cardiology unit, receiving a chronic parenteral nutrition by a Broviac® catheter. The patient presented several thrombosis following iterative catheter replacements. In parallel with superior vena cava plasty, a right intra-atrial Broviac® catheter was placed in the absence of other peripheral venous accesses. This device has a cutaneous exit site to allow for infusion of a hyperosmolar lipid emulsion. Seven days later, a milky liquid was secreted from pericardial/mediastinal redon. A gel lipoprotein electrophoresis of the fluid suggested a preliminary diagnosis of chylopericardium. However, biochemical testing of certain analytes evoked a parenteral nutrition-related pericardial effusion and a possible pseudochyloperitoneum caused by the shearing of a migrated Broviac® in pericardium. The patient, on a fat-free diet, was admitted to the ICU to drain the effusion and reposition the catheter, with success. In the light of new datas on the interference of parenteral lipid emulsions with the lipoproteins gel electrophoresis, we will try to determine whether the apparent presence of chylomicrons in the gel would be the sign of a lesion of the lymphatic system, or rather the result of a contamination by artificial chylomicron in the lipid emulsion, if not the sign of contaminated blood. In our article, we highlight several considerations in identifying and confirming cases of pericardial effusion, such as chylopericardium and parenteral nutrition-related one, as well as points concerning the use of lipid emulsions for pediatric patients with short-bowel syndrome.

Les liquides d'épanchements peuvent renfermer quelques surprises. Nous documentons le cas d'un patient de quatorze mois, hospitalisé en cardiologie, présentant un syndrome de grêle court et recevant une nutrition parentérale au long cours par cathéter Broviac®. Le patient présentait de multiples occlusions veineuses consécutives aux changements itératifs du dispositif. En parallèle d'une plastie de la veine cave supérieure, un Broviac® a été posé en intra-atrial droit devant l'absence d'autres abords veineux périphériques. Ce dispositif comporte un orifice de sortie sous-cutané pour apporter une solution de nutrition hyperosmolaire de type émulsion lipidique. Le liquide recueilli dans les drains péricardiques en post-opératoire est lactescent, particulièrement à partir du septième jour. Le lipidogramme du liquide d'épanchement péricardique semble conclure à la présence de chylomicrons - un chylopéricarde. Cependant, le dosage de certains analytes penche en faveur d'un perfusopéricarde, probablement pseudochyleux, lié au cisaillement du Broviac® dont l'extrémité a migré de l'oreillette droite au péricarde. Le patient, sous régime sans graisses, sans nutrition parentérale, sera réopéré pour drainer l'épanchement et repositionner le cathéter, avec succès. À la lumière de données originales quant à l'interférence des émulsions lipidiques sur le lipidogramme, nous tâcherons de déterminer si l'apparente présence de chylomicrons sur le gel serait le témoin d'une réelle lésion du lymphatique, ou plutôt le fruit d'une contamination par l'émulsion, si ce n'est par le sang. Des considérations au sujet des épanchements péricardiques, dont les chylopéricarde et nutripéricarde, ainsi que sur les émulsions lipidiques pédiatriques dans le contexte du grêle court émailleront ce travail.

Validation of the Performance of A1HPV6, a Triage Blood Test for the Early Diagnosis and Prognosis of SARS-CoV-2 Infection.

BACKGROUND AND AIMS: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. METHODS: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and sex-adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. RESULTS: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97-0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99-0.99; P = .80), like for sensitivity 92% (85-96) vs 94% (88-97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. CONCLUSION: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

Burden of liver disease progression in hospitalized patients with type 2 diabetes mellitus.

BACKGROUND & AIMS: There are uncertainties regarding the burden of liver disease in patients with type 2 diabetes (T2D). Thus, we aimed to quantify the burden of liver disease, identify risk factors, and estimate attributable risks in patients with T2D. METHODS: We measured adjusted hazard ratios of liver disease progression to hepatocellular carcinoma and/or decompensated cirrhosis in a 2010-2020 retrospective, bicentric, longitudinal, cohort of 52,066 hospitalized patients with T2D. RESULTS: Mean age was 64±14 years and 58% were men. Alcohol use disorders accounted for 57% of liver-related complications and were associated with all liver-related risk factors. Non-metabolic liver-related risk factors accounted for 37% of the liver burden. T2D control was not associated with liver disease progression. The incidence (95% CI) of liver-related complications and of competing mortality were 3.9 (3.5-4.3) and 27.8 (26.7-28.9) per 1,000 person-years at risk, respectively. The cumulative incidence of liver disease progression exceeded the cumulative incidence of competing mortality only in the presence of well-identified risk factors of liver disease progression, including alcohol use. The incidence of hepatocellular carcinoma was 0.3 (95% CI 0.1-0.5) per 1,000 person-years in patients with obesity and it increased with age. The adjusted hazard ratios of liver disease progression were 55.7 (40.5-76.6), 3.5 (2.3-5.2), 8.9 (6.9-11.5), and 1.5 (1.1-2.1), for alcohol-related liver disease, alcohol use disorders without alcohol-related liver disease, non-metabolic liver-related risk factors, and obesity, respectively. The attributable fractions of alcohol use disorders, non-metabolic liver-related risk factors, and obesity to the liver burden were 55%, 14%, and 7%, respectively. CONCLUSIONS: In this analysis of data from 2 hospital-based cohorts of patients with T2D, alcohol use disorders, rather than obesity, contributed to most of the liver burden. These results suggest that patients with T2D should be advised to drink minimal amounts of alcohol. LAY SUMMARY: There is uncertainty on the burden of liver-related complications in patients with type 2 diabetes. We studied the risks of liver cancer and complications of liver disease in over 50,000 patients with type 2 diabetes. We found that alcohol was the main factor associated with complications of liver disease. This finding has major implications on the alcohol advice given to patients with type 2 diabetes.

Ex-Vivo Pharmacological Defatting of the Liver: A Review.

The ongoing organ shortage has forced transplant teams to develop alternate sources of liver grafts. In this setting, ex-situ machine perfusion has rapidly developed as a promising tool to assess viability and improve the function of organs from extended criteria donors, including fatty liver grafts. In particular, normothermic machine perfusion represents a powerful tool to test a liver in full 37 °C metabolism and add pharmacological corrections whenever needed. In this context, many pharmacological agents and therapeutics have been tested to induce liver defatting on normothermic machine perfusion with promising results even on human organs. This systematic review makes a comprehensive synthesis on existing pharmacological therapies for liver defatting, with special focus on normothermic liver machine perfusion as an experimental ex-vivo translational model.

Performance of serum apolipoprotein-A1 as a sentinel of Covid-19.

BACKGROUND: Since 1920, a decrease in serum cholesterol has been identified as a marker of severe pneumonia. We have assessed the performance of serum apolipoprotein-A1, the main transporter of HDL-cholesterol, to identify the early spread of coronavirus disease 2019 (Covid-19) in the general population and its diagnostic performance for the Covid-19. METHODS: We compared the daily mean serum apolipoprotein-A1 during the first 34 weeks of 2020 in a population that is routinely followed for a risk of liver fibrosis risk in the USA (212,297 serum) and in France (20,652 serum) in relation to a local increase in confirmed cases, and in comparison to the same period in 2019 (266,976 and 28,452 serum, respectively). We prospectively assessed the sensitivity of this marker in an observational study of 136 consecutive hospitalized cases and retrospectively evaluated its specificity in 7,481 controls representing the general population. RESULTS: The mean serum apolipoprotein-A1 levels in the survey populations began decreasing in January 2020, compared to the same period in 2019. This decrease was highly correlated with the daily increase in confirmed Covid-19 cases in the following 34 weeks, both in France and USA, including the June and mid-July recovery periods in France. Apolipoprotein-A1 at the 1.25 g/L cutoff had a sensitivity of 90.6% (95%CI84.2-95.1) and a specificity of 96.1% (95.7-96.6%) for the diagnosis of Covid-19. The area under the characteristics curve was 0.978 (0.957-0.988), and outperformed haptoglobin and liver function tests. The adjusted risk ratio of apolipoprotein-A1 for survival without transfer to intensive care unit was 5.61 (95%CI 1.02-31.0; P = 0.04). CONCLUSION: Apolipoprotein-A1 could be a sentinel of the pandemic in existing routine surveillance of the general population. NCT01927133, CER-2020-14.

Creatinine clearance after cimetidine administration in a new short procedure: comparison with plasma and renal clearances of iohexol.

BACKGROUND: Creatinine clearance after cimetidine administration (Cim-CreatClr) was once proposed as a method of glomerular filtration rate (GFR) measurement, but has been largely abandoned. We investigated whether a new short procedure for Cim-CreatClr determination could be considered an appropriate method for GFR measurement. METHODS: A 150-min protocol involving oral cimetidine administration was developed to determine Cim-CreatClr. In total, 168 patients underwent simultaneous assessments of creatinine clearance before and after cimetidine administration [basal creatinine clearance (Basal-CreatClr) and Cim-CreatClr, respectively], renal iohexol clearance and plasma iohexol clearance (R-iohexClr and P-iohexClr, respectively). We compared the agreement between the various methods of GFR measurement, using Bland-Altman plots to determine biases, precisions (standard deviation of the biases) and accuracy (proportions of GFR values falling within 10, 15 and 30% of the mean: P10, P15 and P30, respectively). RESULTS: After cimetidine administration, Basal-CreatClr decreased by 19.8% [95% reference limits of agreement (95% LoA): -2.2 to 41.7%]. The bias between Cim-CreatClr and P-iohexClr was -0.6% (95% LoA -26.8 to 28%); the precision was 14.0%; P10, P15 and P30 were 57.1% [95% confidence interval (95% CI) 49.3 to 64.7%], 73.2% (95% CI 65.8 to 79.7%) and 97.0% (95% CI 93.2 to 99.0%), respectively. Due to the positive bias (16.7%; 95% LoA -3.6 to 36.9%) of Cim-CreatClr relative to R-iohexClr, accuracy of Cim-CreatClr relative to R-iohexClr was poor despite a good precision (10.3%). CONCLUSIONS: Our study shows a high level of agreement between Cim-CreatClr and P-iohexClr. These results suggest that this short Cim-CreatClr procedure is a valid method for GFR measurement, which might be useful, in particular, in situations in which P-iohexClr is not suitable or not available.

Transportation and handling of blood samples prior to ammonia measurement in the real life of a large university hospital.

BACKGROUND: Hyperammonemia is neurotoxic and as such can be a medical emergency. Preanalytical factors greatly influence the blood ammonia concentration results. AIMS AND METHODS: Ammonia concentrations measured in the real life setting of a large hospital after pneumatic transport of blood samples and various time periods before centrifugation were compared to those based on the indications of the reagent manufacturer. In the same routine context, the effects of waiting times of centrifuged samples or after plasma storage at -20 °C and -80 °C were determined. RESULTS: Despite the pneumatic transport, the lead times for sample arrival to the lab were even longer than those recommended for their complete handling until ammonia assay. Ammonia concentration results were not affected by the pneumatic transport of blood samples and by waiting times up to a maximum of 1.75 h before their centrifugation and 1 h after centrifugation. Ammonia stability was superior when plasma was stored at -80 °C. CONCLUSION: Pneumatic transport and sample handling in the routine practice of our lab do not affect ammonia concentration results provided that waiting times are limited to 1.75 h before and 1 h after centrifugation and samples are kept cold. Otherwise, it is better to freeze plasma at -80 °C.

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease.

Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an "HbF per red blood cell (HbF/RBC) threshold" requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF - the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti-Human-HbF antibody, was obtained from non-SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso-occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.

Exome sequencing for diagnosis of congenital hemolytic anemia.

BACKGROUND: Congenital hemolytic anemia constitutes a heterogeneous group of rare genetic disorders of red blood cells. Diagnosis is based on clinical data, family history and phenotypic testing, genetic analyses being usually performed as a late step. In this study, we explored 40 patients with congenital hemolytic anemia by whole exome sequencing: 20 patients with hereditary spherocytosis and 20 patients with unexplained hemolysis. RESULTS: A probable genetic cause of disease was identified in 82.5% of the patients (33/40): 100% of those with suspected hereditary spherocytosis (20/20) and 65% of those with unexplained hemolysis (13/20). We found that several patients carried genetic variations in more than one gene (3/20 in the hereditary spherocytosis group, 6/13 fully elucidated patients in the unexplained hemolysis group), giving a more accurate picture of the genetic complexity of congenital hemolytic anemia. In addition, whole exome sequencing allowed us to identify genetic variants in non-congenital hemolytic anemia genes that explained part of the phenotype in 3 patients. CONCLUSION: The rapid development of next generation sequencing has rendered the genetic study of these diseases much easier and cheaper. Whole exome sequencing in congenital hemolytic anemia could provide a more precise and quicker diagnosis, improve patients' healthcare and probably has to be democratized notably for complex cases.

In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like.

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

Major Role for TRPV1 and InsP3R in PAR2-Elicited Inflammatory Mediator Production in Differentiated Human Keratinocytes.

PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1ß, TNF-α, and TSLP. In this study, we investigated PAR2 calcium signaling and the consequent production of inflammatory mediators in differentiated human primary keratinocytes (DhPKs). Stimulation with the PAR2-activating peptide SLIGKV promoted Ca2+ store depletion in both undifferentiated human primary keratinocytes and DhPKs. SLIGKV-evoked Ca2+ store depletion did not trigger the store-operated Ca2+ entry (i.e., SOCE) through ORAI1 in DhPKs compared with undifferentiated human primary keratinocytes. The inhibition of phospholipase C and the concomitant inhibition of TRPV1 and inositol triphosphate receptor in DhPKs abrogated the SLIGKV-evoked Ca2+ store depletion; NF-κB activity; and the production of inflammatory mediators such as IL-1ß, TNF-α, and TSLP. Taken together, these results indicate a key role for both InsP3R and TRPV1 in Ca2+ internal stores in the PAR2-evoked Ca2+ release and consequent skin inflammation in DhPKs. These findings may provide clues to understanding the pathological role of DhPKs in skin disorders in which PAR2 is known to be involved, such as atopic dermatitis, Netherton syndrome, and psoriasis.

A new tool to test active ingredient using lactic acid in vitro, a help to understand cellular mechanism involved in stinging test: An example using a bacterial polysaccharide (Fucogel® ).

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A soothing sensation of cosmetics or ingredients can be also appreciated through a decrease in stinging score. To predict the soothing sensation of a product before in vivo testing, we developed a model based on an LA test and substance P (SP) release using a co-culture of human keratinocytes and NGF-differentiated PC12 cells. A bacterial fucose-rich polysaccharide present in Fucogel® was evaluated as the soothing molecule in the in vivo stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease in SP release from the two cellular types and in co-cultures without modifying the pH of the medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease in prickling intensity vs the placebo in 19 women between the ages of 21 and 69. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation and limits human volunteers. Moreover, Fucogel® reduced prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic inflammation as showed by our new in vitro stinging test based on SP release.