Laboratory of Excellence GR-Ex

BACKGROUND: Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resultant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is central to the pathogenesis of acute attacks and chronic symptoms in acute hepatic porphyria. Givosiran, an RNA interference therapy, inhibits ALAS1 expression. METHODS: In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned symptomatic patients with acute hepatic porphyria to receive either subcutaneous givosiran (2.5 mg per kilogram of body weight) or placebo monthly for 6 months. The primary end point was the annualized rate of composite porphyria attacks among patients with acute intermittent porphyria, the most common subtype of acute hepatic porphyria. (Composite porphyria attacks resulted in hospitalization, an urgent health care visit, or intravenous administration of hemin at home.) Key secondary end points were levels of ALA and porphobilinogen and the annualized attack rate among patients with acute hepatic porphyria, along with hemin use and daily worst pain scores in patients with acute intermittent porphyria. RESULTS: A total of 94 patients underwent randomization (48 in the givosiran group and 46 in the placebo group). Among the 89 patients with acute intermittent porphyria, the mean annualized attack rate was 3.2 in the givosiran group and 12.5 in the placebo group, representing a 74% lower rate in the givosiran group (P<0.001); the results were similar among the 94 patients with acute hepatic porphyria. Among the patients with acute intermittent porphyria, givosiran led to lower levels of urinary ALA and porphobilinogen, fewer days of hemin use, and better daily scores for pain than placebo. Key adverse events that were observed more frequently in the givosiran group were elevations in serum aminotransferase levels, changes in serum creatinine levels and the estimated glomerular filtration rate, and injection-site reactions. CONCLUSIONS: Among patients with acute intermittent porphyria, those who received givosiran had a significantly lower rate of porphyria attacks and better results for multiple other disease manifestations than those who received placebo. The increased efficacy was accompanied by a higher frequency of hepatic and renal adverse events. (Funded by Alnylam Pharmaceuticals; ENVISION ClinicalTrials.gov number, NCT03338816.).

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

Blood viscosity is an important determinant of local flow characteristics, which exhibits shear thinning behavior: it decreases exponentially with increasing shear rates. Both hematocrit and plasma viscosity influence blood viscosity. The shear thinning property of blood is mainly attributed to red blood cell (RBC) rheological properties. RBC aggregation occurs at low shear rates, and increases blood viscosity and depends on both cellular (RBC aggregability) and plasma factors. Blood flow in the microcirculation is highly dependent on the ability of RBC to deform, but RBC deformability also affects blood flow in the macrocirculation since a loss of deformability causes a rise in blood viscosity. Indeed, any changes in one or several of these parameters may affect blood viscosity differently. Poiseuille's Law predicts that any increase in blood viscosity should cause a rise in vascular resistance. However, blood viscosity, through its effects on wall shear stress, is a key modulator of nitric oxide (NO) production by the endothelial NO-synthase. Indeed, any increase in blood viscosity should promote vasodilation. This is the case in healthy individuals when vascular function is intact and able to adapt to blood rheological strains. However, in sickle cell disease (SCD) vascular function is impaired. In this context, any increase in blood viscosity can promote vaso-occlusive like events. We previously showed that sickle cell patients with high blood viscosity usually have more frequent vaso-occlusive crises than those with low blood viscosity. However, while the deformability of RBC decreases during acute vaso-occlusive events in SCD, patients with the highest RBC deformability at steady-state have a higher risk of developing frequent painful vaso-occlusive crises. This paradox seems to be due to the fact that in SCD RBC with the highest deformability are also the most adherent, which would trigger vaso-occlusion. While acute, intense exercise may increase blood viscosity in healthy individuals, recent works conducted in sickle cell patients have shown that light cycling exercise did not cause dramatic changes in blood rheology. Moreover, regular physical exercise has been shown to decrease blood viscosity in sickle cell mice, which could be beneficial for adequate blood flow and tissue perfusion.

Amyotrophic lateral sclerosis (ALS) is a devastating condition with an estimated mortality of 30,000 patients a year worldwide. The median reported survival time since onset ranges from 24 to 48 months. Riluzole is the only currently approved mildly efficacious treatment. Riluzole received marketing authorization in 1995 in the USA and in 1996 in Europe. In the years that followed, over 60 molecules have been investigated as a possible treatment for ALS. Despite significant research efforts, the overwhelming majority of human clinical trials (CTs) have failed to demonstrate clinical efficacy. In the past year, oral masitinib and intravenous edaravone have emerged as promising new therapeutics with claimed efficacy in CTs in ALS patients. Given their advanced phase of clinical development one may consider these drugs as the most likely near-term additions to the therapeutic arsenal available for patients with ALS. In terms of patient inclusion, CT with masitinib recruited a wider, more representative, less restrictive patient population in comparison to the only successful edaravone CT (edaravone eligibility criteria represents only 18% of masitinib study patients). The present manuscript reviews >50 CTs conducted in the last 20 years since riluzole was first approved. A special emphasis is put on the analysis of existing evidence in support of the clinical efficacy of edaravone and masitinib and the possible implications of an eventual marketing authorisation in the treatment of ALS.

Observers inspected normal, high quality colour displays of everyday visual scenes while their eye movements were recorded. A large display change occurred each time an eye blink occurred. Display changes could either involve "Central Interest" or "Marginal Interest" locations, as determined from descriptions obtained from independent judges in a prior pilot experiment. Visual salience, as determined by luminance, colour, and position of the Central and Marginal Interest changes were equalized.

Ferroptosis is an iron-dependent form of cell death characterized by intracellular lipid peroxide accumulation and redox imbalance. Ferroptosis shows specific biological and morphological features when compared to the other cell death patterns. The loss of lipid peroxide repair activity by glutathione peroxidase 4 (GPX4), the presence of redox-active iron and the oxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids are considered as distinct fingerprints of ferroptosis. Several pathways, including amino acid and iron metabolism, ferritinophagy, cell adhesion, p53, Keap1/Nrf2 and phospholipid biosynthesis, can modify susceptibility to ferroptosis. Through the decades, various diseases, including acute kidney injury; cancer; ischemia-reperfusion injury; and cardiovascular, neurodegenerative and hepatic disorders, have been associated with ferroptosis. In this review, we provide a comprehensive analysis of the main biological and biochemical mechanisms of ferroptosis and an overview of chemicals used as inducers and inhibitors. Then, we report the contribution of ferroptosis to the spectrum of liver diseases, acute or chronic. Finally, we discuss the use of ferroptosis as a therapeutic approach against hepatocellular carcinoma, the most common form of primary liver cancer.

The spleen has a combined function of immune defence and quality control of senescent or altered red cells. It is the first organ injured in sickle cell anaemia (SCA) with evidence of hyposplenism present before 12 months in the majority of children. Repeated splenic vaso-occlusion leads to fibrosis and progressive atrophy of the organ (autosplenectomy), which is generally complete by 5 years in SCA. The precise sequence of pathogenic events leading to hyposplenism is unknown. Splenic injury is generally silent and progressive. It can be clinically overt with acute splenic sequestration of red cells, an unpredictable and life-threatening complication in infants. Splenomegaly, with or without hypersplenism, can also occur and can coexist with loss of function. Hyposplenism increases the susceptibility of SCA children to infection with encapsulated bacteria, which is notably reduced by penicillin prophylaxis and immunization. Whether hyposplenism indirectly increases the risk of vaso-occlusion or other circulatory complications remains to be determined.

The human gastrointestinal tract is inhabited by the largest microbial community within the human body consisting of trillions of microbes called gut microbiota. The normal flora is the site of many physiological functions such as enhancing the host immunity, participating in the nutrient absorption and protecting the body against pathogenic microorganisms. Numerous investigations showed a bidirectional interplay between gut microbiota and many organs within the human body such as the intestines, the lungs, the brain, and the skin. Large body of evidence demonstrated, more than a decade ago, that the gut microbial alteration is a key factor in the pathogenesis of many local and systemic disorders. In this regard, a deep understanding of the mechanisms involved in the gut microbial symbiosis/dysbiosis is crucial for the clinical and health field. We review the most recent studies on the involvement of gut microbiota in the pathogenesis of many diseases. We also elaborate the different strategies used to manipulate the gut microbiota in the prevention and treatment of disorders. The future of medicine is strongly related to the quality of our microbiota. Targeting microbiota dysbiosis will be a huge challenge.

Red blood cells (RBCs) can be cleared from circulation when alterations in their size, shape, and deformability are detected. This function is modulated by the spleen-specific structure of the interendothelial slit (IES). Here, we present a unique physiological framework for development of prognostic markers in RBC diseases by quantifying biophysical limits for RBCs to pass through the IES, using computational simulations based on dissipative particle dynamics. The results show that the spleen selects RBCs for continued circulation based on their geometry, consistent with prior in vivo observations. A companion analysis provides critical bounds relating surface area and volume for healthy RBCs beyond which the RBCs fail the "physical fitness test" to pass through the IES, supporting independent experiments. Our results suggest that the spleen plays an important role in determining distributions of size and shape of healthy RBCs. Because mechanical retention of infected RBC impacts malaria pathogenesis, we studied key biophysical parameters for RBCs infected with Plasmodium falciparum as they cross the IES. In agreement with experimental results, surface area loss of an infected RBC is found to be a more important determinant of splenic retention than its membrane stiffness. The simulations provide insights into the effects of pressure gradient across the IES on RBC retention. By providing quantitative biophysical limits for RBCs to pass through the IES, the narrowest circulatory bottleneck in the spleen, our results offer a broad approach for developing quantitative markers for diseases such as hereditary spherocytosis, thalassemia, and malaria.

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described and is a constitutional inherited bone marrow failure syndrome. Erythroblastopenia is the major characteristic of the disease, which is a model for ribosomal diseases, related to a heterozygous allelic variation in 1 of the 20 ribosomal protein genes of either the small or large ribosomal subunit. The salient feature of classical DBA is a defect in ribosomal RNA maturation that generates nucleolar stress, leading to stabilization of p53 and activation of its targets, resulting in cell-cycle arrest and apoptosis. Although activation of p53 may not explain all aspects of DBA erythroid tropism, involvement of GATA1/HSP70 and globin/heme imbalance, with an excess of the toxic free heme leading to reactive oxygen species production, account for defective erythropoiesis in DBA. Despite significant progress in defining the molecular basis of DBA and increased understanding of the mechanistic basis for DBA pathophysiology, progress in developing new therapeutic options has been limited. However, recent advances in gene therapy, better outcomes with stem cell transplantation, and discoveries of putative new drugs through systematic drug screening using large chemical libraries provide hope for improvement.

Repeated traumatic events induce long-lasting behavioral changes that are key to organism adaptation and that affect cognitive, emotional, and social behaviors. Rodents subjected to repeated instances of aggression develop enduring social aversion and increased anxiety. Such repeated aggressions trigger a stress response, resulting in glucocorticoid release and activation of the ascending dopamine (DA) system. We bred mice with selective inactivation of the gene encoding the glucocorticoid receptor (GR) along the DA pathway, and exposed them to repeated aggressions. GR in dopaminoceptive but not DA-releasing neurons specifically promoted social aversion as well as dopaminergic neurochemical and electrophysiological neuroadaptations. Anxiety and fear memories remained unaffected. Acute inhibition of the activity of DA-releasing neurons fully restored social interaction in socially defeated wild-type mice. Our data suggest a GR-dependent neuronal dichotomy for the regulation of emotional and social behaviors, and clearly implicate GR as a link between stress resiliency and dopaminergic tone.

Halogen atoms have been at the center of many rational medicinal chemistry applications in drug design. While fluorine and chlorine atoms are often added to enhance physicochemical properties, bromine and iodine elements are generally inserted to improve selectivity. Favorable halogen interactions such as halogen bond have been thoroughly studied through quantum mechanics and statistical analyses. Although most of the studies focus on halogen interaction through its σ-hole, hydrogen bonding also has a significant impact. Here, we present an analysis describing the interacting environment of halogen atoms in protein-ligand context. With consideration of structural redundancy in the PDB, tendencies toward specific molecular interactions consideration have been refined and implications for rational drug design with halogens further discussed. Finally, we highlight the moderate occurrence of halogen bonding and present the other roles of halogen in protein-ligand complexes, completing the medicinal chemistry guide to rational halogen interactions.

Sickle cell disease (SCD) is a genetic disease caused by a single mutation in the β-globin gene, leading to the production of an abnormal hemoglobin called hemoglobin S (HbS), which polymerizes under deoxygenation, and induces the sickling of red blood cells (RBCs). Sickled RBCs are very fragile and rigid, and patients consequently become anemic and develop frequent and recurrent vaso-occlusive crises. However, it is now evident that SCD is not only a RBC rheological disease. Accumulating evidence shows that SCD is also characterized by the presence of chronic inflammation and oxidative stress, participating in the development of chronic vasculopathy and several chronic complications. The accumulation of hemoglobin and heme in the plasma, as a consequence of enhanced intravascular hemolysis, decreases nitric oxide bioavailability and enhances the production of reactive oxygen species (ROS). Heme and hemoglobin also represent erythrocytic danger-associated molecular pattern molecules (eDAMPs), which may activate endothelial inflammation through TLR-4 signaling and promote the development of complications, such as acute chest syndrome. It is also suspected that heme may activate the innate immune complement system and stimulate neutrophils to release neutrophil extracellular traps. A large amount of microparticles (MPs) from various cellular origins (platelets, RBCs, white blood cells, endothelial cells) is also released into the plasma of SCD patients and participate in the inflammation and oxidative stress in SCD. In turn, this pro-inflammatory and oxidative stress environment further alters the RBC properties. Increased pro-inflammatory cytokine concentrations promote the activation of RBC NADPH oxidase and, thus, raise the production of intra-erythrocyte ROS. Such enhanced oxidative stress causes deleterious damage to the RBC membrane and further alters the deformability of the cells, modifying their aggregation properties. These RBC rheological alterations have been shown to be associated to specific SCD complications, such as leg ulcers, priapism, and glomerulopathy. Moreover, RBCs positive for the Duffy antigen receptor for chemokines may be very sensitive to various inflammatory molecules that promote RBC dehydration and increase RBC adhesiveness to the vascular wall. In summary, SCD is characterized by a vicious circle between abnormal RBC rheology and inflammation, which modulates the clinical severity of patients.

Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contrast to the low/undetectable hepcidin levels observed in acquired iron deficiency, in patients with Matriptase-2 deficiency, serum hepcidin is inappropriately high for the low iron status and accounts for the absent/delayed response to oral iron treatment. A challenge for the clinicians and pediatricians is the recognition of the disorder among iron deficiency and other microcytic anemias commonly found in pediatric patients. The current treatment of iron refractory iron deficiency anemia is based on parenteral iron administration; in the future, manipulation of the hepcidin pathway with the aim of suppressing it might become an alternative therapeutic approach.

BACKGROUND AND AIMS: Acute hepatic porphyria comprises a group of rare genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long-term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients. APPROACH AND RESULTS: EXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months before the study, patients reported a median (range) of 6 (0-52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a health care facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0-37.0]). Elevated levels of hepatic δ-aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ-aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased health care utilization. CONCLUSIONS: Patients experienced attacks often requiring treatment in a health care facility and/or with hemin, as well as chronic symptoms that adversely influenced day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies.

AIMS: In the context of recent concerns regarding performance enhancing techniques and potential negative health effects of high-level physical activity, data on the long-term outcomes and causes of death in elite endurance cyclists are of particular interest. METHODS AND RESULTS: Characteristics and vital status of all French participants in the Tour de France were collected for the 1947-2012 period. Causes of death were obtained from 1968. Overall and disease-specific mortalities were compared with the French male population using overall and specific standardized mortality ratios (SMRs) with their 95% confidence intervals (CIs). Among the 786 French cyclists who participated at least once between 1947 and 2012, 208 (26%) died by 1 September 2012. Neoplasms and cardiovascular diseases accounted for 61% of deaths. We observed a 41% lower mortality in French cyclists (SMR: 0.59, 95% CI: 0.51-0.68, P < 0.0001), which did not change over time (P = 0.70). It was observed for main mortality causes: for neoplasms (SMR: 0.56; 95% CI: 0.42-0.72, P < 0.0001) and for cardiovascular death (SMR: 0.67; 95% CI: 0.50-0.88, P = 0.004), except mortality related to external causes (SMR: 1.06, 95% CI: 0.71-1.53, P = 0.80). CONCLUSION: We observed a substantially and significantly lower mortality in participants in the Tour de France, compared with the general male population. However, our results do not allow us to assess in detail the balance between positive effects of high-level sports activity and selection of healthy elite athletes, vs. any potential deleterious effects of excessive physical exercise or alleged doping.

INTRODUCTION: Anemia in children is a major public health problem throughout the world. It is often multifactorial, iron deficiency being the most frequent etiology. Consequences are diverse and largely under evaluated. Areas covered: This paper briefly reviews the main causes and focus on the potential consequences of acute and chronic anemia in children. Expert commentary: Anemia in children should never be trivialized. Even if iron deficiency is frequently involved, other potentially life-threatening causes are possible and should be looked for. The exact contribution of anemia to child mortality and morbidity is difficult to assess because of overlapping comorbidities. Chronic anemia may impair growth, cardiac function and cognitive development in infants but other consequences are rather poorly described and should be explored more thoroughly.

Transfusions can be a life-saving treatment of patients with sickle-cell disease (SCD). However, availability of matched units can be limiting because of distinctive blood group polymorphisms in patients of African descent. Development of antibodies against the transfused red blood cells (RBCs), resulting in delayed hemolytic transfusion reactions (DHTRs), can be life-threatening and pose unique challenges for this population with regard to treatment strategies and transfusion management protocols. In cases where the transfused cells and the patient's own RBCs are destroyed, diagnosis of DHTR can be difficult because symptoms may mimic vaso-occlusive crisis, and frequently, antibodies are undetectable. Guidelines are needed for early diagnosis of DHTR because treatment may need to include temporarily withholding any new transfusions to avoid further hemolysis. Also needed are case-control studies to optimally tailor treatments based on the severity of DHTR and develop preventive transfusion strategies for patients at DHTR risk. Here, we will review gaps in knowledge and describe through case studies our recommended approach to prevent alloimmunization and to diagnose and treat symptomatic DHTRs for which complementary mechanistic studies to understand their pathogenesis are sorely needed.

BACKGROUND: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS. METHODS: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset. RESULTS: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %. CONCLUSIONS: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.