Testing the efficacy and safety of BIO101, for the prevention of respiratory deterioration, in patients with COVID-19 pneumonia (COVA study): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: As of December, 1st, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia. TRIAL DESIGN: Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive - to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial. PARTICIPANTS: Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days - due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients' participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days. INTERVENTION AND COMPARATOR: Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., 'negative' or 'positive' event). Participants who are officially discharged from hospital care will no longer receive study medication. MAIN OUTCOMES: Primary study endpoint: The proportion of participants with 'negative' events up to 28 days. 'Negative' events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with 'positive' events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A 'positive' event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected. RANDOMIZATION: Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not). BLINDING (MASKING): Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacydata, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1). TRIAL STATUS: The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1st 2020 is ongoing and is anticipated to finish for the whole study by March2021. TRIAL REGISTRATION: The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Neural circuit repair by low-intensity magnetic stimulation requires cellular magnetoreceptors and specific stimulation patterns.

Although electromagnetic brain stimulation is a promising treatment in neurology and psychiatry, clinical outcomes are variable, and underlying mechanisms are ill-defined, which impedes the development of new effective stimulation protocols. Here, we show, in vivo and ex vivo, that repetitive transcranial magnetic stimulation at low-intensity (LI-rTMS) induces axon outgrowth and synaptogenesis to repair a neural circuit. This repair depends on stimulation pattern, with complex biomimetic patterns being particularly effective, and the presence of cryptochrome, a putative magnetoreceptor. Only repair-promoting LI-rTMS patterns up-regulated genes involved in neuronal repair; almost 40% of were cryptochrome targets. Our data open a new framework to understand the mechanisms underlying structural neuroplasticity induced by electromagnetic stimulation. Rather than neuronal activation by induced electric currents, we propose that weak magnetic fields act through cryptochrome to activate cellular signaling cascades. This information opens new routes to optimize electromagnetic stimulation and develop effective treatments for different neurological diseases.

Norepinephrine transporter expression is inversely associated with glycaemic indices: a pilot study in metabolically diverse persons with overweight and obesity.

OBJECTIVE: The objective of this study was to examine the cross-sectional relationship between the expression of norepinephrine transporter (NET), the protein responsible for neuronal uptake-1, and indices of glycaemia and hyperinsulinaemia, in overweight and obese individuals. METHODS: Thirteen non-medicated, non-smoking subjects, aged 58 ± 1 years (mean ± standard error of the mean), body mass index (BMI) 31.4 ± 1.0 kg m-2, with wide-ranging plasma glucose and haemoglobin A1c (HbA1c, range 5.1% to 6.5%) participated. They underwent forearm vein biopsy to access sympathetic nerves for the quantification of NET by Western blot, oral glucose tolerance test (OGTT), euglycaemic hyperinsulinaemic clamp, echocardiography and assessments of whole-body norepinephrine kinetics and muscle sympathetic nerve activity. RESULTS: Norepinephrine transporter expression was inversely associated with fasting plasma glucose (r = -0.62, P = 0.02), glucose area under the curve during OGTT (AUC0-120, r = -0.65, P = 0.02) and HbA1c (r = -0.67, P = 0.01), and positively associated with steady-state glucose utilization during euglycaemic clamp (r = 0.58, P = 0.04). Moreover, NET expression was inversely related to left ventricular posterior wall dimensions (r = -0.64, P = 0.02) and heart rate (r = -0.55, P = 0.05). Indices of hyperinsulinaemia were not associated with NET expression. In stepwise linear regression analysis adjusted for age, body mass index and blood pressure, HbA1c was an independent inverse predictor of NET expression, explaining 45% of its variance. CONCLUSIONS: Hyperglycaemia is associated with reduced peripheral NET expression. Further studies are required to identify the direction of causality.

Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers.

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic ß-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor.

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial function in an experimental model of heart failure in large animals.

Abnormal excitation-contraction coupling is a key pathophysiologic component of heart failure (HF), and at a molecular level reduced expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) is a major contributor. Previous studies in small animals have suggested that restoration of SERCA function is beneficial in HF. Despite this promise, the means by which this information might be translated into potential clinical application remains uncertain. Using a recently established cardiac-directed recirculating method of gene delivery, we administered adeno-associated virus 2 (AAV2)/1SERCA2a to sheep with pacing-induced HF. We explored the effects of differing doses of AAV2/1SERCA2a (low 1 x 10(10) d.r.p.; medium 1 x 10(12) d.r.p. and high 1 x 10(13) d.r.p.) in conjunction with an intra-coronary delivery group (2.5 x 10(13) d.r.p.). At the end of the study, haemodynamic, echocardiographic, histopathologic and molecular biologic assessments were performed. Cardiac recirculation delivery of AAV2/1SERCA2a elicited a dose-dependent improvement in cardiac performance determined by left ventricular pressure analysis, (+d P/d t(max); low dose -220+/-70, P>0.05; medium dose 125+/-53, P<0.05; high dose 287+/-104, P<0.05) and echocardiographically (fractional shortening: low dose -3+/-2, P>0.05; medium dose 1+/-2, P>0.05; high dose 6.5+/-3.9, P<0.05). In addition to favourable haemodynamic effects, brain natriuretic peptide expression was reduced consistent with reversal of the HF molecular phenotype. In contrast, direct intra-coronary infusion did not elicit any effect on ventricular function. As such, AAV2/1SERCA2a elicits favourable functional and molecular actions when delivered in a mechanically targeted manner in an experimental model of HF. These observations lay a platform for potential clinical translation.

BCL-2 counteracts Doppel-induced apoptosis of prion-protein-deficient Purkinje cells in the Ngsk Prnp(0/0) mouse.

The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.

BAX contributes to Doppel-induced apoptosis of prion-protein-deficient Purkinje cells.

Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.

Upstream control of apoptosis by caspase-2 in serum-deprived primary neurons.

During development as well as in pathological situations, neurons that fail to find appropriate targets or neurotrophic factors undergo cell death. Using primary cortical neurons subjected to acute serum-deprivation (SD), we have examined caspases activation, mitochondrial dysfunction and cell death parameters. Among a panel of metabolic, signaling and caspases inhibitors only those able to interfere with caspase-2 like activity protect primary neurons against SD-induced cell death. In situ detection and subcellular fractionation demonstrate a very early activation of cytosolic caspase-2, which controls Bax cleavage, relocalization and mitochondrial membrane permeabilization (MMP). Both z-VDVAD-fmk and a siRNA specific for caspase-2 abolish Bax changes, mitochondrial membranes permeabilization, as well as cytochrome c release-dependent activation of caspase-9/caspase-3, nuclear alterations, phosphatidylserine exposure, neurites dismantling and neuronal death. Hence, caspase-2 is an early checkpoint for apoptosis initiation in primary neurons subjected to serum deprivation.

C-Jun N-terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis.

Understanding the regulation of the apoptotic program in neurons by intracellular pathways is currently a subject of great interest. Recent results suggest that c-Jun N-terminal kinases (JNK), mitogen-activated protein kinases and the transcription factor c-Jun are important regulators of this cell death program in post-mitotic neurons following survival-factor withdrawal. Our study demonstrates that ceramide levels increase upon survival-factor withdrawal in primary cultured cortical neurons. Furthermore, survival-factor withdrawal or addition of exogenous c(2)-ceramide induces JNK pathway activation in these cells. Western blot analyses of JNK and c-Jun using phospho-specific antibodies reveal that JNK and subsequent c-Jun phosphorylation occur hours before the initiation of apoptosis, reflected morphologically by neurite retraction and fragmentation, cell-body shrinkage and chromatin fragmentation. Immunocytochemistry using the same antibodies shows that phospho-JNK are localized in the neurites of control neurons and translocate to the nucleus where phospho-c-Jun concurrently appears upon ceramide-induced apoptosis. To determine if ceramide-induced c-Jun activation is responsible for the induction of the apoptotic program, we performed transient transfections of a dominant negative form of c-Jun, truncated in its transactivation region. Our results show that DNc-Jun partially protects cortical neurons from ceramide-induced apoptosis. Treatment of dominant negative c-Jun-expressing neurons with the pharmacological inhibitor of p38 kinase, SB203580, completely blocked neuronal death. Thus our data show that p38 and JNK/c-Jun pathways cooperate to induce neuronal apoptosis.

Increased ischemia-induced angiogenesis in the staggerer mouse, a mutant of the nuclear receptor Roralpha.

Ror alpha is an orphan nuclear receptor. In homozygous staggerer mutant mice (Ror alpha(sg/sg)), a deletion within the Ror alpha gene leads to an overexpression of inflammatory cytokines. Because inflammation and hypoxia are 2 key stimuli of ischemia-induced angiogenesis, we studied the role of Ror alpha in this setting. Ischemia was induced by ligation of the right femoral artery in C57BL/6 Ror alpha(+/+) and Ror alpha(sg/sg) mice. After 3 and 28 days, angiogenesis was evaluated by microangiography, measurement of capillary density using immunohistochemistry (anti-CD31), and measurement of blood flow by laser Doppler imaging. At day 3, angiographic score and blood flow were similar in Ror alpha(sg/sg) mice and in Ror alpha(+/+) littermates. Conversely, at day 28, Ror alpha(sg/sg) mice showed a significant 2-fold increase in angiographic score and a 3-fold increase in capillary density within the ischemic hindlimb compared with control. Functionally, this coincided with a significant rise in leg perfusion in Ror alpha(sg/sg) mice (0.83+/-0.05 for ischemic/nonischemic leg perfusion ratio) compared with Ror(+/+) mice (0.66+/-0.04, P<0.05). In addition, more extensive angiogenesis in Ror alpha(sg/sg) mice correlated with an increased expression of eNOS protein by 83+/-12% and 71+/-24% at 3 and 28 days, respectively (P<0.05), whereas the level of the antiangiogenic cytokine IL-12 was significantly reduced by 38+/-10% at day 28 (P<0.05). Conversely, no changes in VEGF expression were observed. Our study identifies for the first time a new role for Ror alpha as a potent negative regulator of ischemia-induced angiogenesis.

Temperament related to early-onset substance use: test of a developmental model.

We tested a theoretical model of early-onset substance (tobacco, alcohol, and marijuana) use. A sample of 1,810 public school students was surveyed in sixth grade (M age 11.5 years) and seventh grade. Temperament dimensions were related to substance use, and structural modeling analyses showed indirect effects through self-control constructs. Good self-control had a path to higher academic competence and had direct effects to less peer use and less adolescent substance use; poor self-control had a path to more adolescent life events and more deviant peer affiliations. Academic competence and life events had indirect effects to adolescent substance use, through peer affiliations. Findings from self-report data were corroborated by independent teacher ratings. Effects were also noted for family variables and demographic characteristics. Implications of epigenetic theory for prevention research are discussed.

Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38 phosphorylation and not by the decrease in ERK phosphorylation.

Ceramide, the central molecule of the sphingomyelin pathway, serves as a second messenger for cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis. In this study we show that c2-ceramide induces apoptosis in primary cortical neuron cultures and that this effect correlates with differential modulation of mitogen-activated protein kinase (MAPK) cascades. Phosphorylation of extracellular signal-regulated kinases (ERKs) and their upstream activators MAPK kinases (MEKs), as measured by immunoblotting is rapidly decreased by c2-ceramide. However, the MEK inhibitor PD98059 alone does not induce apoptosis and in combination with c2-ceramide it does not modify c2-ceramide-induced apoptosis. Treatment with c2-ceramide increases p38 and c-Jun N-terminal kinase (JNK) phosphorylation before and during caspase-3 activation. The p38 inhibitor SB203580 partially protects cortical neurons against c2-ceramide-induced apoptosis, implicating the p38 pathway in this process. The c2-ceramide treatment also increases levels of c-jun, c-fos and p53 mRNA in primary cortical neuron cultures, but this is independent of p38 activation. Our study further elucidates the time-courses of MAPK cascade modulation, and of c-jun, c-fos and p53 activation during c2-ceramide-induced neuronal apoptosis. It reveals that one of the activated kinases, p38, is necessary for this apoptosis.

Transgenic mice with neuronal overexpression of bcl-2 gene present navigation disabilities in a water task.

In the CNS, Bcl-2 is an antiapoptotic gene involved in the regulation of neuronal death. Transgenic mice overexpressing the human gene Bcl-2 (Hu-bcl-2 mice) showed delayed acquisition in two tasks requiring them to find a hidden platform starting from either a random or a constant starting location. The same mice were not deficient in another task requiring them to find a visible platform suggesting that the delay observed was not due to motor, visual or motivational deficits in the water. The delay observed in Hu-bcl-2 mice was more important in the random starting test in which the allocentric demand for navigation was stronger. The results suggested that allocentric navigation is particularly sensitive to abnormal CNS maturation following the overexpression of the bcl-2 gene. The specific deficits (motor learning, fear-related behavior and allocentric navigation) observed in Hu-bcl-2 mice suggest that the regulation of developmental neuronal death is crucial for multisensorial learning and emotional behavior.

The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response.

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha.

Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis. Because apolipoprotein (apo) C-III is a major determinant of plasma triglyceride and remnant lipoprotein metabolism, it is important to understand how the expression of this gene is regulated. In the present study, we identified the orphan nuclear receptor RORalpha1 as a regulator of human and mouse apo C-III gene expression. Plasma triglyceride and apo C-III protein concentrations in staggerer (sg/sg) mice, homozygous for a deletion in the RORalpha gene, were significantly lower than in wild type littermates. The lowered plasma apo C-III levels were associated with reduced apo C-III mRNA levels in liver and intestine of sg/sg mice. Transient transfection experiments in human hepatoma HepG2, human colonic CaCO2, and rabbit kidney RK13 cells demonstrated that overexpression of the human RORalpha1 isoform specifically increases human apo C-III promoter activity, indicating that RORalpha1 enhances human apo C-III gene transcription. RORalpha1 response elements were mapped by promoter deletion analysis and gel shift experiments to two AGGTCA half-sites located at positions -83/-78 (within the C3P site) and -23/-18 (downstream of the TATA box) in the human apo C-III promoter, with the -23/-18 site exhibiting the highest binding affinity. Transfection of site-directed mutated constructs in HepG2 cells indicated that the RORalpha1 effect is predominantly mediated by the -23/-18 site. This site is conserved in the mouse apo C-III gene promoter. Moreover, RORalpha binds to the equivalent mouse site and activates constructs containing three copies of the mouse site cloned in front of an heterologous promoter. Taken together, our data identify RORalpha as a transcriptional regulator of apo C-III gene expression, providing a novel, physiological role for RORalpha1 in the regulation of genes controlling triglyceride metabolism.

Tolerance to ischemia and hypoxia is reduced in aged human myocardium.

BACKGROUND: Recovery of cardiac function after cardiac surgery and other interventional cardiac procedures in elderly patients is inferior to that in younger patients, suggesting that the aged myocardium is more sensitive to ischemia and other stresses. Although convincing data from animal studies of senescence now exist, there is a dearth of controlled in vitro studies that examine the specific response of aged human myocardium to the stress of hypoxia or ischemia. OBJECTIVE: We sought to determine the effect of age on the capacity of human atrial trabeculae to recover contractile function after in vitro hypoxic or ischemic stress. METHODS: Atrial pectinate trabeculae were dissected from the tip of 58 right atrial appendages harvested during an operation in patients aged between 34 and 89 years and electrically stimulated at 1 Hz in oxygenated Ringer's solution at 37 degrees C. Tissues experienced 30 minutes of either hypoxia (N(2) and perfusate) or simulated ischemia (humidified N(2) without perfusate) and were returned to normoxia for recovery of function for 30 minutes. Developed force and other contractile variables were determined during each period. RESULTS: Under normoxic conditions, no significant age difference was observed for any contractile function variable. However, after hypoxia, the old (70-89 years) and intermediate age groups (60-69 years) showed reduced recovery of developed force (48.5% +/- 22.2% [n = 11] and 44.9% +/- 19% [n = 12], respectively) compared with that found (66.4% +/- 19.7% [n = 15]) in the younger (34-59 years) group (mean +/- SD, P =.02). Similarly, after simulated ischemia, the groups of 70- to 89-year-old and 60- to 69-year-old subjects showed reduced recovery of developed force (35.7% +/- 17% [n = 5] and 51.1% +/- 11.8% [n = 9], respectively) compared with that found (68.2% +/- 10.4% [n = 6]) in the group of 34- to 59-year-old subjects (P =.01). Multivariable analysis, comparing 20 factors of surgical patient characteristics and recovery of developed force, found that only age (P =.01) and hypertension (P =.01) were predictors of reduced recovery of developed force after either hypoxia or simulated ischemia. CONCLUSIONS: In aged human atrial myocardium, the capacity to recover contractile function after in vitro hypoxia or simulated ischemia is reduced compared with the younger myocardium of mature adults. These findings suggest that enhanced myocardial protective strategies may be indicated for elderly patients undergoing cardiac surgery.