Adjunctive everolimus therapy for tuberous sclerosis complex-associated refractory seizures: Results from the postextension phase of EXIST-3.

OBJECTIVE: Epilepsy is highly prevalent in patients with tuberous sclerosis complex (TSC). Everolimus showed higher efficacy than placebo for seizures in the primary analysis of the EXIST-3 study. Here, we present the long-term outcomes of everolimus at the end of the postextension phase (PEP; data cutoff date: October 25, 2017). METHODS: After completion of the extension phase, patients were invited to continue everolimus in the PEP with everolimus (targeted trough concentration = 5-15 ng/ml, investigator-judged). Efficacy assessments included changes in seizure status during the PEP collected at 12-week intervals as parent/caregiver-reported data through a structured questionnaire. RESULTS: Among 361 patients, 343 entered the extension phase and 249 entered the PEP. After 12 weeks in the PEP, 18.9% (46/244) of patients were seizure-free since the last visit of the extension phase and 64.8% (158/244) had a stable/improved seizure status. At 24 weeks, the corresponding percentages were 18.2% (42/231) and 64.5% (149/231). Among 244 patients, the response rate was 32.8% (80/244) during the 12-week maintenance period of the core phase and 63.9% (156/244) at the end of the extension phase. Of the 149 responders at the end of the extension phase, 70.5% were seizure-free or had stable/improved seizure status. Long-term efficacy data showed persistent responses were observed in 183 of 361 patients (50.7%); 63.9% of these patients had a response that lasted at least 48 weeks. The most frequent Grade 3-4 adverse events (≥2% incidence) reported throughout the study were pneumonia, status epilepticus, seizure, stomatitis, neutropenia, and gastroenteritis. Four patients died during the study. SIGNIFICANCE: The final analysis of EXIST-3 demonstrated the sustained efficacy of everolimus as adjunctive therapy in patients with TSC-associated treatment-refractory seizures, with a tolerable safety profile.

Everolimus long-term use in patients with tuberous sclerosis complex: Four-year update of the EXIST-2 study.

OBJECTIVES: We examined the long-term effects of everolimus in patients with renal angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis. METHODS: Following favorable results from the double-blind core phase of EXIST-2 (NCT00790400), patients were allowed to receive open-label everolimus (extension phase). Patients initially randomly assigned to everolimus continued on the same dose; those who were receiving placebo crossed over to everolimus 10 mg/day. Dose modifications were based on tolerability. The primary end point was angiomyolipoma response rate, defined as a ≥50% reduction from baseline in the sum volume of target renal angiomyolipomas in the absence of new target angiomyolipomas, kidney volume increase of >20% from nadir, and angiomyolipoma-related bleeding grade ≥2. The key secondary end point was safety. RESULTS: Of the 112 patients who received ≥1 dose of everolimus, 58% (95% CI, 48.3% to 67.3%) achieved angiomyolipoma response. Almost all patients (97%) experienced reduction in renal lesion volumes at some point during the study period. Median duration of everolimus exposure was 46.9 months. Sixteen (14.3%) patients experienced angiomyolipoma progression at some point in the study. No angiomyolipoma-related bleeding or nephrectomies were reported. One patient on everolimus underwent embolization for worsening right flank pain. Subependymal giant cell astrocytoma lesion response was achieved in 48% of patients and skin lesion response in 68% of patients. The most common adverse events suspected to be treatment-related were stomatitis (42%), hypercholesterolemia (30.4%), acne (25.9%), aphthous stomatitis and nasopharyngitis (each 21.4%). Ten (8.9%) patients withdrew because of an adverse event. Renal function remained stable, and the frequency of emergent adverse events generally decreased over time. CONCLUSIONS: Everolimus treatment remained safe and effective over approximately 4 years. The overall risk/benefit assessment supports the use of everolimus as a viable treatment option for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00790400.

Adjunctive everolimus therapy for treatment-resistant focal-onset seizures associated with tuberous sclerosis (EXIST-3): a phase 3, randomised, double-blind, placebo-controlled study.

BACKGROUND: Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, has been used for various benign tumours associated with tuberous sclerosis complex. We assessed the efficacy and safety of two trough exposure concentrations of everolimus, 3-7 ng/mL (low exposure) and 9-15 ng/mL (high exposure), compared with placebo as adjunctive therapy for treatment-resistant focal-onset seizures in tuberous sclerosis complex. METHODS: In this phase 3, randomised, double-blind, placebo-controlled study, eligible patients aged 2-65 years with tuberous sclerosis complex and treatment-resistant seizures (≥16 in an 8-week baseline phase) receiving one to three concomitant antiepileptic drugs were recruited from 99 centres across 25 countries. Participants were randomly assigned (1:1:1), via permuted-block randomisation (block size of six) implemented by Interactive Response Technology software, to receive placebo, low-exposure everolimus, or high-exposure everolimus. Randomisation was stratified by age subgroup (<6 years, 6 to <12 years, 12 to <18 years, and ≥18 years). Patients, investigators, site personnel, and the sponsor's study team were masked to treatment allocation. The starting dose of everolimus depended on age, body-surface area, and concomitant use of cytochrome 3A4/P-glycoprotein inducers. Dose adjustments were done to attain target trough ranges during a 6-week titration period, and as needed during a 12-week maintenance period of core phase. Patients or their caregivers recorded events in a seizure diary throughout the study. The primary endpoint was change from baseline in the frequency of seizures during the maintenance period, defined as response rate (the proportion of patients achieving ≥50% reduction in seizure frequency) and median percentage reduction in seizure frequency, in all randomised patients. This study is registered with ClinicalTrials.gov, number NCT01713946. FINDINGS: Between July 3, 2013, and May 29, 2015, 366 patients were enrolled and randomly assigned to placebo (n=119), low-exposure everolimus, (n=117), or high-exposure everolimus (n=130). The response rate was 15·1% with placebo (95% CI 9·2-22·8; 18 patients) compared with 28·2% for low-exposure everolimus (95% CI 20·3-37·3; 33 patients; p=0·0077) and 40·0% for high-exposure everolimus (95% CI 31·5-49·0; 52 patients; p<0·0001). The median percentage reduction in seizure frequency was 14·9% (95% CI 0·1-21·7) with placebo versus 29·3% with low-exposure everolimus (95% CI 18·8-41·9; p=0·0028) and 39·6% with high-exposure everolimus (95% CI 35·0-48·7; p<0·0001). Grade 3 or 4 adverse events occurred in 13 (11%) patients in the placebo group, 21 (18%) in the low-exposure group, and 31 (24%) in the high-exposure group. Serious adverse events were reported in three (3%) patients who received placebo, 16 (14%) who received low-exposure everolimus, and 18 (14%) who received high-exposure everolimus. Adverse events led to treatment discontinuation in two (2%) patients in the placebo group versus six (5%) in the low-exposure group and four (3%) in the high-exposure group. INTERPRETATION: Adjunctive everolimus treatment significantly reduced seizure frequency with a tolerable safety profile compared with placebo in patients with tuberous sclerosis complex and treatment-resistant seizures. FUNDING: Novartis Pharmaceuticals Corporation.

Everolimus for renal angiomyolipoma in patients with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis: extension of a randomized controlled trial.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors are recommended as first-line treatment of renal angiomyolipoma associated with tuberous sclerosis complex (TSC) or sporadic lymphangioleiomyomatosis (sporadic LAM), but follow-up is limited. Longer term efficacy and tolerability data from a Phase 3, double-blind, placebo-controlled trial are presented. METHODS: Following favorable results from the primary analysis (data cutoff 30 June 2011) of the EXIST-2 trial, patients still receiving study treatment were allowed to enter an open-label extension. Everolimus was initiated at 10 mg once daily and titrated based on tolerability. The primary outcome was angiomyolipoma response rate (≥ 50% reduction from baseline in target lesion volumes). Safety was a secondary endpoint. RESULTS: As of the cutoff date (1 May 2013), 112 patients had received everolimus, and the response rate in 107 patients with angiomyolipoma (median duration of medication exposure of 28.9 months) was 54%. The proportion of patients achieving angiomyolipoma reductions of ≥ 30% and ≥ 50% increased over time, reaching 81.6% (62/76) and 64.5% (49/76), respectively, by Week 96. No everolimus-treated patients experienced renal bleeding. The long-term safety profile was consistent with previous reports; adverse events (AEs) were mostly Grade 1/2, and there were no new safety issues. The frequency of emerging AEs and severe AEs lessened over time. CONCLUSIONS: Longer term everolimus treatment appeared safe and effective in patients with TSC- or sporadic LAM-associated renal angiomyolipoma not requiring surgical intervention. Continued reduction in angiomyolipoma volume was demonstrated, and there was no angiomyolipoma-related bleeding; AEs were predictable and generally manageable. TRIAL REGISTRATION: clinicaltrialsgov identifier: NCT00790400 (http://clinicaltrials.gov/ct2/show/NCT00790400).

Everolimus for subependymal giant cell astrocytoma: 5-year final analysis.

OBJECTIVE: To analyze the cumulative efficacy and safety of everolimus in treating subependymal giant cell astrocytomas (SEGA) associated with tuberous sclerosis complex (TSC) from an open-label phase II study (NCT00411619). Updated data became available from the conclusion of the extension phase and are presented in this ≥5-year analysis. METHODS: Patients aged ≥ 3 years with a definite diagnosis of TSC and increasing SEGA lesion size (≥2 magnetic resonance imaging scans) received everolimus starting at 3mg/m(2) /day (titrated to target blood trough levels of 5-15ng/ml). The primary efficacy endpoint was reduction from baseline in primary SEGA volume. RESULTS: As of the study completion date (January 28, 2014), 22 of 28 (78.6%) initially enrolled patients finished the study per protocol. Median (range) duration of exposure to everolimus was 67.8 (4.7-83.2) months; 12 (52.2%) and 14 (60.9%) of 23 patients experienced SEGA volume reductions of ≥50% and ≥30% relative to baseline, respectively, after 60 months of treatment. The proportion of patients experiencing daily seizures was reduced from 7 of 26 (26.9%) patients at baseline to 2 of 18 (11.1%) patients at month 60. Most commonly reported adverse events (AEs) were upper respiratory tract infection and stomatitis of mostly grade 1 or 2 severity. No patient discontinued treatment due to AEs. The frequency of emergence of most AEs decreased over the course of the study. INTERPRETATION: Everolimus continues to demonstrate a sustained effect on SEGA tumor reduction over ≥5 years of treatment. Everolimus remained well-tolerated, and no new safety concerns were noted.

Forearm ischemia decreases endothelial colony-forming cell angiogenic potential.

BACKGROUND AIMS: Circulating endothelial progenitor cells and especially endothelial colony-forming cells (ECFCs) are promising candidate cells for endothelial regenerative medicine of ischemic diseases, but the conditions for an optimal collection from adult blood must be improved. METHODS: On the basis of a recently reported vascular niche of ECFCs, we hypothesized that a local ischemia could trigger ECFC mobilization from the vascular wall into peripheral blood to optimize their collection for autologous implantation in critical leg ischemia. Because the target population with critical leg ischemia is composed of elderly patients in whom a vascular impairment has been documented, we also analyzed the impact of aging on ECFC mobilization and vascular integrity. RESULTS: After having defined optimized ECFC culture conditions, we studied the effect of forearm ischemia on ECFC numbers and functions in 26 healthy volunteers (13 volunteers ages 20-30-years old versus 13 volunteers ages 60-70 years old). The results show that forearm ischemia induced an efficient local ischemia and a normal endothelial response but did not mobilize ECFCs regardless of the age group. Moreover, we report an alteration of angiogenic properties of ECFCs obtained after forearm ischemia, in vitro as well as in vivo in a hindlimb ischemia murine model. This impaired ECFC angiogenic potential was not associated with a quantitative modification of the circulating endothelial compartment. CONCLUSIONS: The procedure of local ischemia, although reulting in a preserved endothelial reactivity, did not mobilize ECFCs but altered their angiogenic potential.

Effect of contrasted sodium diets on the pharmacokinetics and pharmacodynamic effects of renin-angiotensin system blockers.

Dietary sodium, the main determinant of the pharmacodynamic response to renin-angiotensin system blockade, influences the pharmacokinetics of various cardiovascular drugs. We compared the effect of contrasted sodium diets on the pharmacokinetics of single oral doses of 8 mg candesartan cilexetil, 160 mg valsartan, 10 mg ramipril, and 50 mg atenolol administered to 64 (16 per group) normotensive male subjects randomly assigned to sodium depletion (SD) or sodium repletion (SR) in a crossover study. Pharmacodynamic response was assessed as the increase in plasma renin concentration for renin-angiotensin system blockers and electrocardiographic changes in PR interval duration for atenolol. The area under the curve (AUC) for plasma candesartan and atenolol concentrations was significantly lower for SR than for SD (respective ratios of AUC0-∞: 0.74; [90% CI, 0.66-0.82] and 0.69 [90% CI, 0.54-0.88], respectively), indicating a lack of bioequivalence between SR and SD. SR did not affect the pharmacokinetics of valsartan or ramipril. The increase in plasma renin concentration with the 3 renin-angiotensin system blockers was 10 times lower during the SR than the SD period. In the multiple regression analysis, the AUC0-24 of plasma drug concentration explained <1% and 21% of the variance of the AUC0-24 of delta plasma renin concentration for candesartan (P=0.8882/P=0.0368) during the SR and SD periods, respectively. The atenolol-induced lengthening of PR interval was fully reversed by SR. Thus, sodium balance modulates the pharmacokinetics of candesartan cilexetil and atenolol, with measurable effects on the selected pharmacodynamic end points.

Sequential comparison of aldosterone synthase inhibition and mineralocorticoid blockade in patients with primary aldosteronism.

OBJECTIVE: We compared the effects of aldosterone synthase inhibition with LCI699 with those of mineralocorticoid receptor blockade in patients with primary aldosteronism. METHODS: After a 2-week placebo run-in, 14 patients with primary aldosteronism received oral LCI699 (0.5 mg b.i.d.) from day 1 to 14, LCI699 (1 mg b.i.d.) from day 15 to 29, and placebo from day 29 to 36. From day 36 to day 66, patients were treated with eplerenone (50 mg b.i.d., up-titrated to 100 mg b.i.d. in 12/14 patients), in addition to their previous antihypertensive treatment, which was maintained unchanged. RESULTS: Eplerenone significantly decreased more 24-h ambulatory SBP on day 66 than LCI699 on day 29 and the difference between the two treatment was -5.34 [95% confidence interval (CI) -10.30; -0.38)] mmHg (P = 0.027). Plasma potassium concentration achieved on eplerenone (4.30 ±â€Š0.45 mmol/l) was significantly greater than on LCI699 (3.89 ±â€Š0.35 mmol/l; P = 0.009). The increase in plasma renin concentration was significantly greater after eplerenone [+131% (range 61; 231)] than on LCI699 on day 29 [+39% (range 5; 86); P = 0.023]. LCI699 markedly decreased plasma aldosterone concentration by 75% (range -84;-63), whereas eplerenone markedly increased this concentration, from day 36, by 89% (range 40;154; P < 0.0001 vs. day 29). CONCLUSION: In patients with primary aldosteronism, the effects on blood pressure and plasma potassium and renin concentrations of 4 weeks of eplerenone treatment (50-100 mg b.i.d.) were more marked than those of 4 weeks of LCI699 treatment (0.5-1 mg b.i.d.). These two drugs had opposite effects on plasma aldosterone concentration.

Imaging work-up for screening of paraganglioma and pheochromocytoma in SDHx mutation carriers: a multicenter prospective study from the PGL.EVA Investigators.

CONTEXT: Recommendations have not been established concerning imaging to screen SDHx mutation carriers for paraganglioma and pheochromocytoma. OBJECTIVE: Our objective was to compare the performance of gadolinium-enhanced magnetic resonance angiography, contrast-enhanced computed tomography, and [(123)I]metaiodo-benzylguanidine and somatostatin receptor scintigraphies for detecting head and neck and thoracic-abdominal-pelvic paragangliomas in SDHx mutation carriers. DESIGN AND SETTING: We conducted a prospective, multicenter study from June 2005 to December 2009 at 23 French medical centers. PATIENTS: A total of 238 index cases or relatives carrying mutations in SDHD, SDHB, or SDHC genes were included. INTERVENTION: Images obtained by each technique were analyzed blind, without knowledge of results from other tests, first in each local center and then centrally. MAIN OUTCOME MEASURES: We evaluated sensitivity, specificity, and likelihood ratios for individual and combinations of tests, the gold standard being the consensus of an expert committee. RESULTS: Two hundred two tumors were diagnosed in 96 subjects. At local assessment, the sensitivity of anatomical imaging for detecting all tumors was higher (85.7%) than that of both scintigraphic techniques (42.7% for [(123)I]metaiodo-benzylguanidine and 69.5% for somatostatin receptor scintigraphy), except for thoracic localizations where somatostatin receptor scintigraphy was more sensitive (61.5 vs. 46.2% for anatomical imaging and 30.8% for [(123)I]metaiodo-benzylguanidine scintigraphy). The best diagnostic performance during local assessment was obtained by combining anatomical imaging tests and somatostatin receptor scintigraphy (sensitivity 91.7%). Central assessment significantly increased the sensitivity (98.6%) of tests in combination. CONCLUSIONS: In routine practice, the imaging work-up for screening SDHx mutation carriers should include thoraco-abdomino-pelvic computed tomography, head and neck magnetic angiography, and somatostatin receptor scintigraphy. Expert centralized image assessment is recommended.

Sequential nephron blockade versus sequential renin-angiotensin system blockade in resistant hypertension: a prospective, randomized, open blinded endpoint study.

OBJECTIVE: To compare two drug regimens to treat resistant hypertension. METHODS: In a prospective, randomized, open blinded endpoint study, 167 patients with mean baseline daytime ambulatory blood pressure 135 mmHg or more and/or 85 mmHg or more, despite 4 weeks' treatment with irbesartan 300 mg/day, hydrochlorothiazide 12.5 mg/day and amlodipine 5 mg/day, were randomized to sequential nephron blockade (group 1, n = 85) or sequential renin-angiotensin system blockade (group 2, n = 82). First, spironolactone 25 mg/day in group 1 or ramipril 5 mg/day in group 2 were added for 4 weeks. Treatment was increased at weeks 4, 8 or 10 if home blood pressure was 135 mmHg or more and/or 85 mmHg or more by sequentially administering furosemide 20 mg/day, furosemide 40 mg/day and amiloride 5 mg/day in group 1, or ramipril 10 mg/day, bisoprolol 5 mg/day and bisoprolol 10 mg/day in group 2. The primary endpoint was change in systolic daytime ambulatory blood pressure at week 12. RESULTS: At week 12, the mean between-group difference in daytime ambulatory blood pressure was 10/4 mmHg (95% confidence interval: 7-14/2-7; P < 0.001/P = 0.0014) in favour of the group 1. The blood pressure goal (daytime ambulatory blood pressure <135/85 mmHg) was achieved in 58% in the group 1 and 20% in the group 2 (P < 0.0001). Discontinuation for drug-related adverse events was low (group 1, n = 7; group 2, n = 6). CONCLUSION: In patients with resistant hypertension, sequential nephron blockade induces a large and well tolerated reduction in blood pressure via a progressive increase in sodium depletion, and is more effective than sequential renin-angiotensin system blockade.

Bosentan effects in hypoxic pulmonary vasoconstriction: Preliminary study in subjects with or without high altitude pulmonary edema-history.

Hypoxia-induced pulmonary vasoconstriction in patients with a medical history of high-altitude pulmonary edema (HAPE) may involve activation of the endothelin-1 (ET-1) pathway. We, therefore, compared the effect of the ETA/ETB receptor antagonist, bosentan, on pulmonary artery systolic pressure (PASP) in healthy subjects with (HS: HAPE subjects, n=5) or without a HAPE-history (CS: Control subjects, n=10). A double-blind, placebo-controlled, randomized, crossover design was performed in order to study the effects on PASP of a single oral dose of bosentan (250 mg) after 90 min exposure to normobaric hypoxia (FiO(2) =0.12). In normoxia, PASP, evaluated by echocardiography, was 23.4±2.7 mmHg in CS and 28±5.8 mmHg in HS (NS). During the placebo period, hypoxia induced a significant decrease in SaO(2), PaO(2) and PCO(2) and increase in pH in both CS and HS. Pulmonary arterial systolic pressure was also significantly increased (+8.5±5.0 mmHg in CS; +13.4±3.1 mmHg in HS) and reached significantly higher levels in HS than in CS (P=0.02). Bosentan significantly but similarly blunted the hypoxia-induced increase in PASP in both CS (Bosentan: 27.0±3.3 mmHg; placebo: 32.1±3.5 mmHg; P<0.01) and HS (Bosentan: 35.0±2.9 mmHg; placebo: 41.4±7.6 mmHg; P<0.05), (CS 5.2±5.3 vs. HS -6.4±5.2 mmHg, NS). Bosentan did not have a major effect on the hypoxia-induced changes in blood gas, or on cardiac output (CO) and systemic blood pressure (SBP), which were not modified by hypoxia. Plasma ET-1 in hypoxia during the bosentan period was 2.8 times higher than during for both CS and HS. A single oral dose of bosentan similarly blunted the hypoxia-induced increase in PASP both in healthy and HAPE-susceptible subjects, without altering CO or SBP.

Immune response to human embryonic stem cell-derived cardiac progenitors and adipose-derived stromal cells.

Transplantation of allogeneic human embryonic stem cell-derived cardiac progenitors triggers an immune response. We assessed whether this response could be modulated by the concomitant use of adipose-derived stromal cells (ADSC). Peripheral blood mononuclear cells were collected from 40 patients with coronary artery disease (CAD) and nine healthy controls. Cardiac progenitors (CD15(+) Mesp1(+)) were generated as already reported from the I6 cell line treated with bone morphogenetic protein (BMP)-2. Adipose-derived stromal cells were obtained from abdominal dermolipectomies. We assessed the proliferative response of peripheral lymphocytes from patients and controls to cardiac progenitors cultured on a monolayer of ADSC, to allogeneic lymphocytes in mixed lymphocyte culture and to the T cell mitogen phytohemaglutin A in presence or absence of ADSC. Cardiac progenitors cultured on a monolayer of ADSC triggered a proliferation of lymphocytes from both patients and controls albeit lower than that induced by allogeneic lymphocytes. When cultured alone, ADSC did not induce any proliferation of allogeneic lymphocytes. When added to cultures of lymphocytes, ADSC significantly inhibited the alloantigen or mitogen-induced proliferative response. Compared to healthy controls, lymphocytes from patients presenting CAD expressed a decreased proliferative capacity, in particular to mitogen-induced stimulation. Adipose-derived stromal cells express an immunomodulatory effect that limits both alloantigen and mitogen-induced lymphocyte responses. Furthermore, lymphocytes from patients with CAD are low responders to conventional stimuli, possibly because of their age and disease-associated treatment regimens. We propose that, in combination, these factors may limit the in vivo immunogenicity of cardiac progenitors co-implanted with ADSC in patients with CAD.

Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections.

AIMS: Intramyocardial injections of cells can damage tissue and enhance dissociation-induced cell death. We assessed whether epicardial delivery of cell sheets could overcome these issues in a rat model of chronic myocardial infarction. METHODS AND RESULTS: Eighty-two rats that had undergone coronary ligation and simultaneous harvest of fat tissue to yield the adipose-derived stromal cell (ADSC) fraction were randomized 1 month after infarction to receive injections of either control medium (n= 24) or 10 × 10(6) autologous ADSC (n= 37) or the epicardial deposit, onto the infarcted area, of a trilayered ADSC sheet (10 × 10(6), n= 21) prepared by culturing cells on temperature-sensitive dishes. Some treated rats received green fluorescent protein labelled ADSC. Survival, function, and cell engraftment were blindly assessed after 2 months. Prior to implantation, cell sheets and suspended cells were assessed for the expression of extracellular matrix constituents and molecules involved in angiogenesis and cardiac remodelling. The survival rate of rats receiving the cell sheets was significantly higher than after cell injections (73 vs. 41%, P = 0.01). This correlated with the absence of left ventricular (LV) remodelling in the cell sheet group, as end-diastolic volume only increased by 2.8% compared with baseline [95% confidence interval (CI): -18.7%; +30.0%, P = 0.81] vs. increases of 25.9% (-0.4%; +59.2%, P = 0.05) and 51.2% (+18.6%; +92.8, P = 0.001) in the cell and medium injection groups, respectively. Sheets also resulted in a greater cell engraftment possibly related to the greater expression of extracellular matrix constituents. CONCLUSION: The better preservation of LV geometry afforded by ADSC sheets is associated with increased survival and engraftment, which supports the concept of an epicardial delivery of cell-seeded biomaterials.

Exhaled nitric oxide and clinical phenotypes of childhood asthma.

Whether exhaled NO helps to identify a specific phenotype of asthmatic patients remains debated. Our aims were to evaluate whether exhaled NO (FENO(0.05)) is independently associated (1) with underlying pathophysiological characteristics of asthma such as airway tone (bronchodilator response) and airway inflammation (inhaled corticosteroid [ICS]-dependant inflammation), and (2) with clinical phenotypes of asthma.We performed multivariate (exhaled NO as dependent variable) and k-means cluster analyses in a population of 169 asthmatic children (age ± SD: 10.5 ± 2.6 years) recruited in a monocenter cohort that was characterized in a cross-sectional design using 28 parameters describing potentially different asthma domains: atopy, environment (tobacco), control, exacerbations, treatment (inhaled corticosteroid and long-acting bronchodilator agonist), and lung function (airway architecture and tone). Two subject-related characteristics (height and atopy) and two disease-related characteristics (bronchodilator response and ICS dose > 200 µg/d) explained 36% of exhaled NO variance. Nine domains were isolated using principal component analysis. Four clusters were further identified: cluster 1 (47%): boys, unexposed to tobacco, with well-controlled asthma; cluster 2 (26%): girls, unexposed to tobacco, with well-controlled asthma; cluster 3 (6%): girls or boys, unexposed to tobacco, with uncontrolled asthma associated with increased airway tone, and cluster 4 (21%): girls or boys, exposed to parental smoking, with small airway to lung size ratio and uncontrolled asthma. FENO(0.05) was not different in these four clusters.In conclusion, FENO(0.05) is independently linked to two pathophysiological characteristics of asthma (ICS-dependant inflammation and bronchomotor tone) but does not help to identify a clinically relevant phenotype of asthmatic children.

Antinatriuretic effect of vasopressin in humans is amiloride sensitive, thus ENaC dependent.

BACKGROUND AND OBJECTIVES: Acute infusion of the potent V2 receptor agonist 1-desamino-8-d-arginine vasopressin (dDAVP) reduces sodium excretion in humans, through an effect attributed to the stimulation of the amiloride sensitive epithelial sodium channel, ENaC, in ex vivo/in vivo experiments. We investigated in humans whether the antinatriuretic effect of dDAVP is sensitive to amiloride, a specific blocker of ENaC. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Forty-eight healthy normotensive adult men were assigned to a high Na/low K (250/40 mmol/d) diet, to suppress aldosterone secretion. dDAVP (4-µg intravenous bolus followed by 4 µg over 2 hours) was administrated before and after a 7-day administration of 20 mg/d amiloride. Urine and blood samples were collected before and at the end of the dDAVP infusion, to measure Na, K, creatinine, and osmolality concentrations. RESULTS: dDAVP alone decreased the urinary flow rate by 75% and the sodium excretion rate by 19% despite an increase in creatinine clearance by 38 ml/min. Potassium excretion rate was unchanged and the urinary Na/K ratio decreased by 18%. Seven-day amiloride administration had no effect on the dDAVP-induced decrease in the urinary flow rate (-71%) nor on the dDAVP-induced increase in creatinine clearance (+35 ml/min), but it fully prevented the dDAVP-induced decrease in both urinary sodium excretion (+1%) and urinary Na/K ratio (+21%). CONCLUSIONS: The antinatriuretic effect of dDAVP in humans is amiloride sensitive, and thus is related to the stimulatory effect on ENaC-mediated sodium reabsorption. This test provides a new tool to investigate ENaC function in a clinical setting.

A decrease of regulatory T cells correlates with overall survival after sunitinib-based antiangiogenic therapy in metastatic renal cancer patients.

Sunitinib, an antiangiogenic molecule, is one of the first-line standard of care in the treatment of patients with metastatic renal cell carcinoma. However, it only benefits to a subgroup of patients and no predictive markers of sunitinib efficacy have been identified. Twenty-eight metastatic renal cell carcinomas were treated with sunitinib-based therapy and another subgroup of 7 primary renal cell cancer patients were also treated by sunitinib in a neoadjuvant trial. Measurements of CD3+CD4+CD25(hi) Foxp3+ regulatory T cells, an immunosuppressive cell population, were performed before and after each cycle of treatment in blood and tumor in a prospective study. We observed a decrease in the number of peripheral blood Foxp3+ regulatory T cells after each cycle of sunitinib-based therapy. The overall survival was significantly longer in patients showing a decrease in the number of Foxp3+ regulatory T cells after 2 or 3 cycles of treatment (P<0.05). The decrease in the number of regulatory T cells positively correlated with their number at baseline (P<0.01), but not with modification of tumor volume defined by Response Evaluation Criteria in Solid Tumors criteria. The clinical relevance of these results was also supported by an intratumoral decrease of regulatory T cells in 5 out of 7 patients treated by sunitinib in a neoadjuvant trial. Our study represents the first work reporting that the measurement of regulatory T cells may have a predictive value on antiangiogenic response. Antiangiogenic therapy also reversed immunosuppression in the tumor microenvironment which provides novel argument in human to favor its combination with immunotherapy.

Aldosterone synthase inhibition with LCI699: a proof-of-concept study in patients with primary aldosteronism.

We report the first administration of an orally active aldosterone synthase inhibitor, LCI699, to 14 patients with primary aldosteronism. After a 2-week placebo run-in, patients received oral LCI699 (0.5 mg BID) for 2 weeks, LCI699 (1.0 mg BID) for 2 weeks, and placebo for 1 week. We assessed changes in hormone concentrations, plasma potassium levels, and 24-hour ambulatory systolic blood pressure and safety. The supine plasma aldosterone concentration decreased from 540 pmol/L (95% CI: 394 to 739 pmol/L) to 171 pmol/L (95% CI: 128 to 230 pmol/L) after 0.5 mg of LCI699 (-68%; P<0.0001) and to 133 pmol/L (95% CI: 100 to 177 pmol/L) after 1.0 mg of LCI699 (-75%; P<0.0001). Plasma 11-deoxycorticosterone concentrations increased by 710% after 0.5 mg of LCI699 (P<0.0001) and by 1427% after 1.0 mg of LCI699 (P<0.0001). The plasma potassium concentration increased from 3.27±0.31 to 4.03±0.33 mmol/L (P<0.0001) after only 1 week on 0.5 mg of LCI699. Twenty-four-hour ambulatory systolic blood pressure decreased by -4.1 mm Hg (95% CI: -8.1 to -0.1 mm Hg) after 4 weeks of treatment (P=0.046). Basal plasma cortisol concentrations remained unchanged, whereas plasma adrenocorticotropic hormone concentrations increased by 35% after 0.5 mg of LCI699 (P=0.08) and by 113% after 1.0 mg of LCI699 (P<0.0001), and the plasma cortisol response to an adrenocorticotropic hormone test was blunted. All of the variables except plasma 11-deoxycorticosterone concentration returned to initial levels after the placebo. LCI699 was well tolerated. In conclusion, the administration of LCI699, up to 1.0 mg BID, effectively and safely inhibits aldosterone synthase in patients with primary aldosteronism. This 4-week treatment corrected the hypokalemia and mildly decreased blood pressure. The effects on the glucocorticoid axis were consistent with a latent inhibition of cortisol synthesis.

Composite cell sheets: a further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells.

BACKGROUND: The safety and efficacy of myocardial regeneration using embryonic stem cells are limited by the risk of teratoma and the high rate of cell death. METHODS AND RESULTS: To address these issues, we developed a composite construct made of a sheet of adipose tissue-derived stroma cells and embryonic stem cell-derived cardiac progenitors. Ten Rhesus monkeys underwent a transient coronary artery occlusion followed, 2 weeks later, by the open-chest delivery of the composite cell sheet over the infarcted area or a sham operation. The sheet was made of adipose tissue-derived stroma cells grown from a biopsy of autologous adipose tissue and cultured onto temperature-responsive dishes. Allogeneic Rhesus embryonic stem cells were committed to a cardiac lineage and immunomagnetically sorted to yield SSEA-1(+) cardiac progenitors, which were then deposited onto the cell sheet. Cyclosporine was given for 2 months until the animals were euthanized. Preimplantation studies showed that the SSEA-1(+) progenitors expressed cardiac markers and had lost pluripotency. After 2 months, there was no teratoma in any of the 5 cell-treated monkeys. Analysis of >1500 histological sections showed that the SSEA-1(+) cardiac progenitors had differentiated into cardiomyocytes, as evidenced by immunofluorescence and real-time polymerase chain reaction. There were also a robust engraftment of autologous adipose tissue-derived stroma cells and increased angiogenesis compared with the sham animals. CONCLUSIONS: These data collected in a clinically relevant nonhuman primate model show that developmentally restricted SSEA-1(+) cardiac progenitors appear to be safe and highlight the benefit of the epicardial delivery of a construct harboring cells with a cardiomyogenic differentiation potential and cells providing them the necessary trophic support.

Thromboangiitis obliterans and endothelial function.

BACKGROUND: Endothelial dysfunction may be involved in the pathophysiology of thromboangiitis obliterans (TAO). This study compares endothelial function and large artery stiffness between 10 TAO patients assessed during an exacerbation phase and 10 age- and sex-matched control subjects. MATERIAL AND METHODS: Flow-mediated vasodilation after gradual hand skin heating (from 28 degrees C to 44 degrees C) and endothelium-independent vasodilation after sublingual administration of 150 microg glyceryl trinitrate (GTN) were studied using a high-resolution echotracking system to simultaneously measure the brachial artery (BA) diameter and changes in wall shear stress. Aortic stiffness was assessed by carotid-to-femoral pulse wave velocity. RESULTS: The baseline BA diameter was significantly smaller in TAO patients (3599 +/- 668 microm) than in control subjects (4114 +/- 671, P = 0.04). Hand warming caused a linear increase in shear stress accompanied by a linear increase in BA diameter as a function of increasing temperature for TAO patients and control subjects. There was no significant difference between the two groups [relative increase in BA diameter: + 9.3% (-0.1 to 11.5) vs. + 4.8% (3.0 to 8.1), respectively; P = 0.63]. The slope of the BA diameter vs. shear stress relationship did not significantly differ between the two groups. The relative increase in the BA diameter after GTN was significantly greater in TAO patients than in controls [+ 30.8% (28.6 to 33.6) vs. + 16.2% (12.6 to 21.9) respectively; P = 0.02]. Finally, TAO patients had greater aortic stiffness than control subjects (9.81 +/- 1.72 m s(-1) vs. 7.82 +/- 0.84 m s(-1) respectively; P = 0.0052). CONCLUSIONS: Acute TAO is characterised by vasoconstriction and increased aortic stiffness in the absence of changes in flow-mediated dilation.

Comparison of endothelial biomarkers according to reversibility of pulmonary hypertension secondary to congenital heart disease.

The reversibility of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is strongly associated with the degree of intimal proliferation, vessel narrowing, and number of circulating endothelial cells (CECs). Circulating endothelial cells may arise from either endothelial damage or accelerated turnover during vessel remodeling, but nothing is known about endothelial microparticles (EMPs) and other biomarkers reflecting endothelial alterations. This study aimed to document endothelial markers further according to the irreversibility of PAH secondary to CHD. The study investigated soluble markers of endothelial damage or activation (thrombomodulin, soluble endothelial protein C receptor, and soluble E-selectin), inflammation (interleukin-6), and angiogenic cytokine levels [vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)] in 26 patients with CHD, 16 with reversible PAH (median age, 2 years) and 10 with irreversible PAH (median age, 9 years). Endothelial activation/apoptosis was evaluated by measuring EMP levels. Plasma procoagulant activity also was measured. The results show that the levels of soluble markers indicating endothelial activation were not predictors of PAH irreversibility. Lower levels of PlGF were observed in reversible compared with irreversible PAH but were not associated with the CEC level, the mean pulmonary artery pressure (mPAP), or age. No significant difference in procoagulant activity or EMP level was found between irreversible and reversible PAH. Among a large panel of biomarkers reflecting endothelial activation, regeneration, and injury, the high CEC levels previously described proved to be the only marker allowing discrimination between reversible and irreversible PAH secondary to CHD.