Prophylaxis with a recombinant factor VIII Fc in hemophilia A: long-term follow-up on joint health, efficacy, and safety from phase 3 studies in children and adults.

Background: Recurrent joint bleeds are a major cause of morbidity in severe hemophilia. Prophylaxis with efmoroctocog alfa (a recombinant factor VIII Fc fusion protein, [rFVIIIFc]) has demonstrated benefits beyond bleed control, including joint health maintenance. Objectives: To assess long-term efficacy and safety of rFVIIIFc prophylaxis in severe hemophilia A in phase 3 pivotal (A-LONG/Kids A-LONG) and extension (ASPIRE) studies. Methods: Longitudinal analysis included pooled data from A-LONG/Kids A-LONG and ASPIRE. Subgroup analyses investigated outcomes in modified Hemophilia Joint Health Score or Hemophilia Joint Health Score and target joints in subjects with 4 to 5 years follow-up on individualized prophylaxis (IP), and those with the highest annualized bleeding rate (ABR) quartile during Year 1 of IP. Results: Overall, rFVIIIFc consumption remained stable and low ABRs were maintained, with a median treatment duration of 4.2/3.4 years in subjects from A-LONG/Kids A-LONG, respectively. Median overall ABR also remained low (1.0-2.0) in subjects on IP for 4 to 5 years. Sustained improvements in modified Hemophilia Joint Health Score or Hemophilia Joint Health Score were demonstrated over a median follow-up of 3.7 years. In subjects from A-LONG/Kids A-LONG, 99.6% (n = 234)/100% (n = 9) of evaluable baseline target joints were resolved, with no recurrence in 95%/100% of target joints. In IP subjects within the highest ABR quartile in Year 1, continued improvements were observed over a median follow-up of 4.3 years in ABR and joint health, without increased factor consumption. No inhibitors or treatment-related serious adverse events were reported. Conclusion: Previously treated subjects of all ages receiving long-term prophylaxis with rFVIIIFc had sustained clinical benefits, including improved joint health and low ABR.

Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

Post hoc longitudinal assessment of the efficacy and safety of recombinant factor IX Fc fusion protein in hemophilia B.

Long-term efficacy and safety of the extended half-life recombinant factor IX Fc fusion protein (rFIXFc) has been established among previously treated patients with severe hemophilia B in 2 phase 3 trials (B-LONG [#NCT01027364] and Kids B-LONG [#NCT01440946]) and a long-term extension study (B-YOND [#NCT01425723]). In this study, we report post hoc analyses of pooled longitudinal data for up to 6.5 years for rFIXFc prophylaxis. In the B-LONG study, subjects ≥12 years received weekly dose-adjusted prophylaxis (WP; starting dose, 50 IU/kg), individualized interval-adjusted prophylaxis (IP; initially, 100 IU/kg every 10 days), or on-demand dosing. In the Kids B-LONG study, subjects <12 years received 50 to 60 IU/kg every 7 days, adjusted as needed. In the B-YOND study, subjects received WP (20-100 IU/kg every 7 days), IP (100 IU/kg every 8-16 days), modified prophylaxis, or on-demand dosing; switching between treatment groups was permitted. A total of 123 subjects from B-LONG and 30 from Kids B-LONG study were included, of whom 93 and 27, respectively, enrolled in the B-YOND study. The median cumulative duration of treatment was 3.63 years (range, 0.003-6.48 years) in B-LONG/B-YOND and 2.88 years (range, 0.30-4.80 years) in Kids B-LONG/B-YOND group. Annualized bleed rates (ABRs) remained low, annualized factor consumption remained stable, and adherence remained high throughout treatment. Low ABRs were also maintained in subjects with dosing intervals ≥14 days or with target joints at baseline. Complete resolution of evaluable target joints and no recurrence in 90.2% of baseline target joints during follow-up were observed. rFIXFc prophylaxis was associated with sustained clinical benefits, including long-term bleed prevention and target joint resolution, for severe hemophilia B.

Recombinant factor VIII Fc fusion protein for first-time immune tolerance induction: final results of the verITI-8 study.

Inhibitor development remains a major challenge in factor VIII (FVIII) replacement therapy. verITI-8 is the first prospective study of a recombinant FVIII Fc fusion protein (rFVIIIFc; efmoroctocog alfa) for first-time immune tolerance induction (ITI) in males with severe hemophilia A and high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL). In this single-arm, open-label, multicenter study, screening was followed by ITI (rFVIIIFc 200 IU/kg per day until tolerization or maximum of 48 weeks). Those who achieved ITI success entered a tapering period, returning to standard prophylaxis, and then entered follow-up. Primary end point was time to tolerization with rFVIIIFc defined by inhibitor titer <0.6 BU/mL, incremental recovery (IR) ≥66% of expected IR (IR ≥1.32 IU/dL per IU/kg), and half-life (t½) ≥7 hours within 48 weeks. Sixteen patients received ≥1 rFVIIIFc dose. Twelve (75%), 11 (69%), and 10 patients (63%), respectively, achieved negative inhibitor titers, an IR ≥66%, and a t½ ≥7 hours (ie, tolerance) within 48 weeks. Median times in weeks to achieve these markers of success were 7.4 (interquartile range [IQR], 2.2-17.8), 6.8 (IQR, 5.4-22.4), and 11.7 (IQR, 9.8-26.2), respectively. All patients experienced ≥1 treatment-emergent adverse event (TEAE), and 1 reported ≥1 related TEAE (injection site pain). Nine patients experienced ≥1 treatment-emergent serious AE. No thrombotic events, discontinuations because of AEs, or deaths were reported during the study. As the first extended half-life rFVIII with prospective data in ITI, rFVIIIFc offered short time to tolerization with durable responses in almost two-thirds of patients and was well tolerated. This trial was registered at www.clinicaltrials.gov as #NCT03093480.

The Yin-Yang of the Green Fluorescent Protein: Impact on Saccharomyces cerevisiae stress resistance.

Although fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang).

Genome-Wide Association Study and Gene-Based Analysis of Participants With Hemophilia A and Inhibitors in the My Life, Our Future Research Repository.

Introduction: Up to 30% of individuals with hemophilia A develop inhibitors to replacement factor VIII (FVIII), rendering the treatment ineffective. The underlying mechanism of inhibitor development remains poorly understood. The My Life, Our Future Research Repository (MLOF RR) has gathered F8 and F9 mutational information, phenotypic data, and biological material from over 11,000 participants with hemophilia A (HA) and B as well as carriers enrolled across US hemophilia treatment centers, including over 5,000 whole-genome sequences. Identifying genes associated with inhibitors may contribute to our understanding of why certain patients develop those neutralizing antibodies. Aim and Methods: Here, we performed a genome-wide association study and gene-based analyses to identify genes associated with inhibitors in participants with HA from the MLOF RR. Results: We identify a genome-wide significant association within the human leukocyte antigen (HLA) locus in participants with HA with F8 intronic inversions. HLA typing revealed independent associations with the HLA alleles major histocompatibility complex, class II, DR beta 1 (HLA DRB1*15:01) and major histocompatibility complex, class II, DQ beta 1 (DQB1*03:03). Variant aggregation tests further identified low-frequency variants within GRID2IP (glutamate receptor, ionotropic, delta 2 [GRID2] interacting protein 1) significantly associated with inhibitors. Conclusion: Overall, our study confirms the association of DRB1*15:01 with FVIII inhibitors and identifies a novel association of DQB1*03:03 in individuals with HA carrying intronic inversions of F8. In addition, our results implicate GRID2IP, encoding GRID2-interacting protein, with the development of inhibitors, and suggest an unrecognized role of this gene in autoimmunity.

A Case Study of Long-Range Rotary Wing Critical Care Transport in the Battlefield Environment.

Military medical evacuation continues to grow both in distance and transport times. With the need for long-range transport of greater than 2 hours, crews are having to manage critical care patients for longer trips. This case study evaluates one specific event in which long-range transport of a sick noncombat patient required an enroute critical care team. Medical electronics and other equipment require special attention. Oxygen bottles and batteries for medical devices become the limiting factor in transport from point to point. Having to juggle multiple data streams requires prioritization and reassessment of interventions. Using the mnemonic "bottles, bags, batteries, battlefield environment" keeps the transport paramedic and enroute care nurse on track to effectively deliver the patient to the next level of care. Consideration should be given to such mnemonics for long critical care transports.

Increased xerotolerance of Saccharomyces cerevisiae during an osmotic pressure ramp over several generations.

Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; aw ) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing-to-destruction) at the rate of 1.5 MPa day-1 from 1.38 to 58.5 MPa (0.990-0.635 aw ). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 aw ) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 aw ). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 aw ) showed a greater cultivability than glycerol-shocked cells after a subsequent shock at 200 MPa (0.234 aw ) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38-10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.

Recombinant Factor VIII Fc Inhibits B Cell Activation via Engagement of the FcγRIIB Receptor.

The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is a major complication of hemophilia A treatment. The sole clinical therapy to restore FVIII tolerance in patients with inhibitors remains immune tolerance induction (ITI) which is expensive, difficult to administer and not always successful. Although not fully understood, the mechanism of ITI is thought to rely on inhibition of FVIII-specific B cells (1). Its efficacy might therefore be improved through more aggressive B cell suppression. FcγRIIB is an inhibitory Fc receptor that down-regulates B cell signaling when cross-linked with the B cell receptor (BCR). We sought to investigate if recombinant FVIII Fc (rFVIIIFc), an Fc fusion molecule composed of FVIII and the Fc region of immunoglobulin G1 (IgG1) (2), is able to inhibit B cell activation more readily than FVIII. rFVIIIFc was able to bind FVIII-exposed and naïve B cells from hemophilia A mice as well as a FVIII-specific murine B cell hybridoma line (413 cells). An anti-FcγRIIB antibody and FVIII inhibited binding, suggesting that rFVIIIFc is able to interact with both FcγRIIB and the BCR. Furthermore, incubation of B cells from FVIII-exposed mice and 413 cells with rFVIIIFc resulted in increased phosphorylation of SH-2 containing inositol 5-phosphatase (SHIP) when compared to FVIII. B cells from FVIII-exposed hemophilia A mice also exhibited decreased extracellular signal-regulated kinase (ERK) phosphorylation when exposed to rFVIIIFc. These differences were absent in B cells from naïve, non-FVIII exposed hemophilic mice suggesting an antigen-dependent effect. Finally, rFVIIIFc was able to inhibit B cell calcium flux induced by anti-Ig F(ab)2. Our results therefore indicate that rFVIIIFc is able to crosslink FcγRIIB and the BCR of FVIII-specific B cells, causing inhibitory signaling in these cells.

Recombinant factor VIII Fc fusion protein drives regulatory macrophage polarization.

The main complication of replacement therapy with factor in hemophilia A (HemA) is the formation of inhibitors (neutralizing anti-factor VIII [FVIII] antibodies) in ∼30% of severe HemA patients. Because these inhibitors render replacement FVIII treatment essentially ineffective, preventing or eliminating them is of top priority in disease management. The extended half-life recombinant FVIII Fc fusion protein (rFVIIIFc) is an approved therapy for HemA patients. In addition, it has been reported that rFVIIIFc may induce tolerance to FVIII more readily than FVIII alone in HemA patients that have developed inhibitors. Given that the immunoglobulin G1 Fc region has the potential to interact with immune cells expressing Fc receptors (FcRs) and thereby affect the immune response to rFVIII, we investigated how human macrophages, expressing both FcRs and receptors reported to bind FVIII, respond to rFVIIIFc. We show herein that rFVIIIFc, but not rFVIII, uniquely skews macrophages toward an alternatively activated regulatory phenotype. rFVIIIFc initiates signaling events that result in morphological changes, as well as a specific gene expression and metabolic profile that is characteristic of the regulatory type Mox/M2-like macrophages. Further, these changes are dependent on rFVIIIFc-FcR interactions. Our findings elucidate mechanisms of potential immunomodulatory properties of rFVIIIFc.

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives.

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.

Evaluation of the toxicology and pharmacokinetics of recombinant factor VIII Fc fusion protein in animals.

INTRODUCTION: Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a novel recombinant factor VIII with a prolonged half-life, developed for the treatment of hemophilia A. Studies that evaluated the toxicological effects of rFVIIIFc in 2 pharmacologically relevant species, cynomolgus monkeys and Sprague Dawley rats, are reported here. MATERIALS AND METHODS: In repeat-dose toxicology studies, rats and monkeys received 0, 50, 250, or 1000 IU/kg rFVIIIFc every other day for 4 weeks. In a high-dose tolerance study, monkeys received 1 rFVIIIFc dose of 3000, 10,000, or 20,000 IU/kg. Evaluations included in-life observations, laboratory and post-mortem evaluations, pharmacokinetics, and local tolerance. Allometric scaling, using data from 4 animal species and humans, was used to evaluate the relationship between animal and human pharmacokinetics. RESULTS: rFVIIIFc was well tolerated with no adverse toxicological findings directly attributable to rFVIIIFc. As expected, antibodies to this fully human protein developed in rats and monkeys in a time-dependent fashion following repeated dosing, leading to increased clearance in both species. There were no local reactions (infusion site) or evidence of thrombosis at high doses in rats and monkeys. Allometric scaling demonstrated more rapid clearance in small animals compared with humans and a volume of distribution (steady state) proportional to body weight across species, suggesting that animal pharmacokinetics are predictive of human pharmacokinetics. CONCLUSIONS: Repeated doses of rFVIIIFc in 2 relevant animal species and high doses of rFVIIIFc in monkeys were well tolerated. These results supported the clinical safety of rFVIIIFc observed in phase 1/2a and phase 3 clinical trials.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

Fully human monoclonal antibody inhibitors of the neonatal fc receptor reduce circulating IgG in non-human primates.

The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.

Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane.

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains. Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable.

Evaluation of the toxicology, pharmacokinetics, and local tolerance of recombinant factor IX Fc fusion protein in animals.

INTRODUCTION: Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. MATERIALS AND METHODS: Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. RESULTS: There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. CONCLUSIONS: rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics.

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn-Fc interaction can generate longer-lasting and more effective therapeutics.

Ozone affects ascorbate and glutathione biosynthesis as well as amino acid contents in three Euramerican poplar genotypes.

Ozone is an air pollutant that causes oxidative stress by generation of reactive oxygen species (ROS) within the leaf. The capacity to detoxify ROS and repair ROS-induced damage may contribute to ozone tolerance. Ascorbate and glutathione are known to be key players in detoxification. Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three Euramerican poplar genotypes (Populus deltoides Bartr. × Populus nigra L.) differing in ozone sensitivity. Total ascorbate and glutathione contents were increased in response to ozone in all genotypes, with the most resistant genotype (Carpaccio) showing an increase of up to 70%. Reduced ascorbate (ASA) concentration at least doubled in the two most resistant genotypes (Carpaccio and Cima), whereas the most sensitive genotype (Robusta) seemed unable to regenerate ASA from oxidized ascorbate (DHA), leading to an increase of 80% of the oxidized form. Increased ascorbate (ASA + DHA) content correlated with the increase in gene expression in its biosynthetic pathway, especially the putative gene of GDP-l-galactose phosphorylase VTC2. Increased cysteine availability combined with increased expression of γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) genes allows higher glutathione biosynthesis in response to ozone, particularly in Carpaccio. In addition, ozone caused a remobilization of amino acids with a decreased pool of total amino acids and an increase of Cys and putrescine, especially in Carpaccio. In addition, the expression of genes encoding threonine aldolase was strongly induced only in the most tolerant genotype, Carpaccio. Reduced ascorbate levels could partly explain the sensitivity to ozone for Robusta but not for Cima. Reduced ascorbate level alone is not sufficient to account for ozone tolerance in poplar, and it is necessary to consider several other factors including glutathione content.

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes.

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H(+) /Ca(2+) -antiports (CAX1 and CAX3 homologs), ß-carbonic anhydrases (ßCA1 and ßCA4) and proton H(+) -ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H(+) /Ca(2+) -antiports.