The Catholic University of Korea St. Vincent's Hospital

BACKGROUND: Patients with diffuse large B-cell lymphoma that is refractory to primary and second-line therapies or that has relapsed after stem-cell transplantation have a poor prognosis. The chimeric antigen receptor (CAR) T-cell therapy tisagenlecleucel targets and eliminates CD19-expressing B cells and showed efficacy against B-cell lymphomas in a single-center, phase 2a study. METHODS: We conducted an international, phase 2, pivotal study of centrally manufactured tisagenlecleucel involving adult patients with relapsed or refractory diffuse large B-cell lymphoma who were ineligible for or had disease progression after autologous hematopoietic stem-cell transplantation. The primary end point was the best overall response rate (i.e., the percentage of patients who had a complete or partial response), as judged by an independent review committee. RESULTS: A total of 93 patients received an infusion and were included in the evaluation of efficacy. The median time from infusion to data cutoff was 14 months (range, 0.1 to 26). The best overall response rate was 52% (95% confidence interval, 41 to 62); 40% of the patients had complete responses, and 12% had partial responses. Response rates were consistent across prognostic subgroups. At 12 months after the initial response, the rate of relapse-free survival was estimated to be 65% (79% among patients with a complete response). The most common grade 3 or 4 adverse events of special interest included cytokine release syndrome (22%), neurologic events (12%), cytopenias lasting more than 28 days (32%), infections (20%), and febrile neutropenia (14%). Three patients died from disease progression within 30 days after infusion. No deaths were attributed to tisagenlecleucel, cytokine release syndrome, or cerebral edema. No differences between response groups in tumor expression of CD19 or immune checkpoint-related proteins were found. CONCLUSIONS: In this international study of CAR T-cell therapy in relapsed or refractory diffuse large B-cell lymphoma in adults, high rates of durable responses were produced with the use of tisagenlecleucel. (Funded by Novartis; JULIET ClinicalTrials.gov number, NCT02445248 .).

BACKGROUND: Restenosis after coronary stenting necessitates repeated percutaneous or surgical revascularization procedures. The delivery of paclitaxel to the site of vascular injury may reduce the incidence of neointimal hyperplasia and restenosis. METHODS: At 73 U.S. centers, we enrolled 1314 patients who were receiving a stent in a single, previously untreated coronary-artery stenosis (vessel diameter, 2.5 to 3.75 mm; lesion length, 10 to 28 mm) in a prospective, randomized, double-blind study. A total of 652 patients were randomly assigned to receive a bare-metal stent, and 662 to receive an identical-appearing, slow-release, polymer-based, paclitaxel-eluting stent. Angiographic follow-up was prespecified at nine months in 732 patients. RESULTS: In terms of base-line characteristics, the two groups were well matched. Diabetes mellitus was present in 24.2 percent of patients; the mean reference-vessel diameter was 2.75 mm, and the mean lesion length was 13.4 mm. A mean of 1.08 stents (length, 21.8 mm) were implanted per patient. The rate of ischemia-driven target-vessel revascularization at nine months was reduced from 12.0 percent with the implantation of a bare-metal stent to 4.7 percent with the implantation of a paclitaxel-eluting stent (relative risk, 0.39; 95 percent confidence interval, 0.26 to 0.59; P<0.001). Target-lesion revascularization was required in 3.0 percent of the group that received a paclitaxel-eluting stent, as compared with 11.3 percent of the group that received a bare-metal stent (relative risk, 0.27; 95 percent confidence interval, 0.16 to 0.43; P<0.001). The rate of angiographic restenosis was reduced from 26.6 percent to 7.9 percent with the paclitaxel-eluting stent (relative risk, 0.30; 95 percent confidence interval, 0.19 to 0.46; P<0.001). The nine-month composite rates of death from cardiac causes or myocardial infarction (4.7 percent and 4.3 percent, respectively) and stent thrombosis (0.6 percent and 0.8 percent, respectively) were similar in the group that received a paclitaxel-eluting stent and the group that received a bare-metal stent. CONCLUSIONS: As compared with bare-metal stents, the slow-release, polymer-based, paclitaxel-eluting stent is safe and markedly reduces the rates of clinical and angiographic restenosis at nine months.

BACKGROUND: In patients with chronic lymphoid leukemia (CLL) or small lymphocytic lymphoma (SLL), a short duration of response to therapy or adverse cytogenetic abnormalities are associated with a poor outcome. We evaluated the efficacy of ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase, in patients at risk for a poor outcome. METHODS: In this multicenter, open-label, phase 3 study, we randomly assigned 391 patients with relapsed or refractory CLL or SLL to receive daily ibrutinib or the anti-CD20 antibody ofatumumab. The primary end point was the duration of progression-free survival, with the duration of overall survival and the overall response rate as secondary end points. RESULTS: At a median follow-up of 9.4 months, ibrutinib significantly improved progression-free survival; the median duration was not reached in the ibrutinib group (with a rate of progression-free survival of 88% at 6 months), as compared with a median of 8.1 months in the ofatumumab group (hazard ratio for progression or death in the ibrutinib group, 0.22; P<0.001). Ibrutinib also significantly improved overall survival (hazard ratio for death, 0.43; P=0.005). At 12 months, the overall survival rate was 90% in the ibrutinib group and 81% in the ofatumumab group. The overall response rate was significantly higher in the ibrutinib group than in the ofatumumab group (42.6% vs. 4.1%, P<0.001). An additional 20% of ibrutinib-treated patients had a partial response with lymphocytosis. Similar effects were observed regardless of whether patients had a chromosome 17p13.1 deletion or resistance to purine analogues. The most frequent nonhematologic adverse events were diarrhea, fatigue, pyrexia, and nausea in the ibrutinib group and fatigue, infusion-related reactions, and cough in the ofatumumab group. CONCLUSIONS: Ibrutinib, as compared with ofatumumab, significantly improved progression-free survival, overall survival, and response rate among patients with previously treated CLL or SLL. (Funded by Pharmacyclics and Janssen; RESONATE ClinicalTrials.gov number, NCT01578707.).

BACKGROUND: Enzalutamide, an androgen-receptor inhibitor, has been associated with improved overall survival in men with castration-resistant prostate cancer. It is not known whether adding enzalutamide to testosterone suppression, with or without early docetaxel, will improve survival in men with metastatic, hormone-sensitive prostate cancer. METHODS: In this open-label, randomized, phase 3 trial, we assigned patients to receive testosterone suppression plus either open-label enzalutamide or a standard nonsteroidal antiandrogen therapy (standard-care group). The primary end point was overall survival. Secondary end points included progression-free survival as determined by the prostate-specific antigen (PSA) level, clinical progression-free survival, and adverse events. RESULTS: A total of 1125 men underwent randomization; the median follow-up was 34 months. There were 102 deaths in the enzalutamide group and 143 deaths in the standard-care group (hazard ratio, 0.67; 95% confidence interval [CI], 0.52 to 0.86; P = 0.002). Kaplan-Meier estimates of overall survival at 3 years were 80% (based on 94 events) in the enzalutamide group and 72% (based on 130 events) in the standard-care group. Better results with enzalutamide were also seen in PSA progression-free survival (174 and 333 events, respectively; hazard ratio, 0.39; P<0.001) and in clinical progression-free survival (167 and 320 events, respectively; hazard ratio, 0.40; P<0.001). Treatment discontinuation due to adverse events was more frequent in the enzalutamide group than in the standard-care group (33 events and 14 events, respectively). Fatigue was more common in the enzalutamide group; seizures occurred in 7 patients in the enzalutamide group (1%) and in no patients in the standard-care group. CONCLUSIONS: Enzalutamide was associated with significantly longer progression-free and overall survival than standard care in men with metastatic, hormone-sensitive prostate cancer receiving testosterone suppression. The enzalutamide group had a higher incidence of seizures and other toxic effects, especially among those treated with early docetaxel. (Funded by Astellas Scientific and Medical Affairs and others; ENZAMET (ANZUP 1304) ANZCTR number, ACTRN12614000110684; ClinicalTrials.gov number, NCT02446405; and EU Clinical Trials Register number, 2014-003190-42.).

BACKGROUND: Aspirin is a well-established therapy for the secondary prevention of cardiovascular events. However, its role in the primary prevention of cardiovascular disease is unclear, especially in older persons, who have an increased risk. METHODS: From 2010 through 2014, we enrolled community-dwelling men and women in Australia and the United States who were 70 years of age or older (or ≥65 years of age among blacks and Hispanics in the United States) and did not have cardiovascular disease, dementia, or disability. Participants were randomly assigned to receive 100 mg of enteric-coated aspirin or placebo. The primary end point was a composite of death, dementia, or persistent physical disability; results for this end point are reported in another article in the Journal. Secondary end points included major hemorrhage and cardiovascular disease (defined as fatal coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal stroke, or hospitalization for heart failure). RESULTS: Of the 19,114 persons who were enrolled in the trial, 9525 were assigned to receive aspirin and 9589 to receive placebo. After a median of 4.7 years of follow-up, the rate of cardiovascular disease was 10.7 events per 1000 person-years in the aspirin group and 11.3 events per 1000 person-years in the placebo group (hazard ratio, 0.95; 95% confidence interval [CI], 0.83 to 1.08). The rate of major hemorrhage was 8.6 events per 1000 person-years and 6.2 events per 1000 person-years, respectively (hazard ratio, 1.38; 95% CI, 1.18 to 1.62; P<0.001). CONCLUSIONS: The use of low-dose aspirin as a primary prevention strategy in older adults resulted in a significantly higher risk of major hemorrhage and did not result in a significantly lower risk of cardiovascular disease than placebo. (Funded by the National Institute on Aging and others; ASPREE ClinicalTrials.gov number, NCT01038583 .).

BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

This collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

BACKGROUND: In phase 2 trials, treatment with the combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor velpatasvir resulted in high rates of sustained virologic response in patients chronically infected with hepatitis C virus (HCV) genotype 2 or 3. METHODS: We conducted two randomized, phase 3, open-label studies involving patients who had received previous treatment for HCV genotype 2 or 3 and those who had not received such treatment, including patients with compensated cirrhosis. In one trial, patients with HCV genotype 2 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir, in a once-daily, fixed-dose combination tablet (134 patients), or sofosbuvir plus weight-based ribavirin (132 patients) for 12 weeks. In a second trial, patients with HCV genotype 3 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir for 12 weeks (277 patients) or sofosbuvir-ribavirin for 24 weeks (275 patients). The primary end point for the two trials was a sustained virologic response at 12 weeks after the end of therapy. RESULTS: Among patients with HCV genotype 2, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 99% (95% confidence interval [CI], 96 to 100), which was superior to the rate of 94% (95% CI, 88 to 97) in the sofosbuvir-ribavirin group (P=0.02). Among patients with HCV genotype 3, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 95% (95% CI, 92 to 98), which was superior to the rate of 80% (95% CI, 75 to 85) in the sofosbuvir-ribavirin group (P<0.001). The most common adverse events in the two studies were fatigue, headache, nausea, and insomnia. CONCLUSIONS: Among patients with HCV genotype 2 or 3 with or without previous treatment, including those with compensated cirrhosis, 12 weeks of treatment with sofosbuvir-velpatasvir resulted in rates of sustained virologic response that were superior to those with standard treatment with sofosbuvir-ribavirin. (Funded by Gilead Sciences; ASTRAL-2 ClinicalTrials.gov number, NCT02220998; and ASTRAL-3, NCT02201953.).

Human electrophysiological (EEG) studies have demonstrated the involvement of alpha band (8- to 14-Hz) oscillations in the anticipatory biasing of attention. In the context of visual spatial attention within bilateral stimulus arrays, alpha has exhibited greater amplitude over parietooccipital cortex contralateral to the hemifield required to be ignored, relative to that measured when the same hemifield is to be attended. Whether this differential effect arises solely from alpha desynchronization (decreases) over the "attending" hemisphere, from synchronization (increases) over the "ignoring" hemisphere, or both, has not been fully resolved. This is because of the confounding effect of externally evoked desynchronization that occurs involuntarily in response to visual cues. Here, bilateral flickering stimuli were presented simultaneously and continuously over entire trial blocks, such that externally evoked alpha desynchronization is equated in precue baseline and postcue intervals. Equivalent random letter sequences were superimposed on the left and right flicker stimuli. Subjects were required to count the presentations of the target letter "X" at the cued hemifield over an 8-s period and ignore the sequence in the opposite hemifield. The data showed significant increases in alpha power over the ignoring hemisphere relative to the precue baseline, observable for both cue directions. A strong attentional bias necessitated by the subjective difficulty in gating the distracting letter sequence is reflected in a large effect size of 2.1 (eta2 = 0.82), measured from the attention x hemisphere interaction. This strongly suggests that alpha synchronization reflects an active attentional suppression mechanism, rather than a passive one reflecting "idling" circuits.

Significant progress has been made in outcome measurement procedures for osteoarthritis (OA) clinical trials, and guidelines have been established by the US Food and Drug Administration, European League Against Rheumatism, the World Health Organization/International League of Associations for Rheumatology, and the Group for the Respect of Ethics and Excellence in Science. However, there remains a need for further international harmonization of measurement procedures used to establish beneficial effects in Phase III clinical trials. A key objective of the OMERACT III conference was to establish a core set of outcome measures for future phase III clinical trials. During the conference, using a combination of discussion and polling procedures, a consensus was reached by at least 90% of participants that the following 4 domains should be evaluated in future phase III trials of knee, hip, and hand OA: pain, physical function, patient global assessment, and, for studies of one year or longer, joint imaging (using standardized methods for taking and rating radiographs, or any demonstrably superior imaging technique). These evidence based preferences, achieved with a high degree of consensus, establish an international standard for future phase III trials and will also facilitate metaanalysis and Cochrane Collaborative Project goals.

BACKGROUND: Adjuvant therapy with an aromatase inhibitor improves outcomes, as compared with tamoxifen, in postmenopausal women with hormone-receptor-positive breast cancer. METHODS: In two phase 3 trials, we randomly assigned premenopausal women with hormone-receptor-positive early breast cancer to the aromatase inhibitor exemestane plus ovarian suppression or tamoxifen plus ovarian suppression for a period of 5 years. Suppression of ovarian estrogen production was achieved with the use of the gonadotropin-releasing-hormone agonist triptorelin, oophorectomy, or ovarian irradiation. The primary analysis combined data from 4690 patients in the two trials. RESULTS: After a median follow-up of 68 months, disease-free survival at 5 years was 91.1% in the exemestane-ovarian suppression group and 87.3% in the tamoxifen-ovarian suppression group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.72; 95% confidence interval [CI], 0.60 to 0.85; P<0.001). The rate of freedom from breast cancer at 5 years was 92.8% in the exemestane-ovarian suppression group, as compared with 88.8% in the tamoxifen-ovarian suppression group (hazard ratio for recurrence, 0.66; 95% CI, 0.55 to 0.80; P<0.001). With 194 deaths (4.1% of the patients), overall survival did not differ significantly between the two groups (hazard ratio for death in the exemestane-ovarian suppression group, 1.14; 95% CI, 0.86 to 1.51; P=0.37). Selected adverse events of grade 3 or 4 were reported for 30.6% of the patients in the exemestane-ovarian suppression group and 29.4% of those in the tamoxifen-ovarian suppression group, with profiles similar to those for postmenopausal women. CONCLUSIONS: In premenopausal women with hormone-receptor-positive early breast cancer, adjuvant treatment with exemestane plus ovarian suppression, as compared with tamoxifen plus ovarian suppression, significantly reduced recurrence. (Funded by Pfizer and others; TEXT and SOFT ClinicalTrials.gov numbers, NCT00066703 and NCT00066690, respectively.).

BACKGROUND: Suppression of ovarian estrogen production reduces the recurrence of hormone-receptor-positive early breast cancer in premenopausal women, but its value when added to tamoxifen is uncertain. METHODS: We randomly assigned 3066 premenopausal women, stratified according to prior receipt or nonreceipt of chemotherapy, to receive 5 years of tamoxifen, tamoxifen plus ovarian suppression, or exemestane plus ovarian suppression. The primary analysis tested the hypothesis that tamoxifen plus ovarian suppression would improve disease-free survival, as compared with tamoxifen alone. In the primary analysis, 46.7% of the patients had not received chemotherapy previously, and 53.3% had received chemotherapy and remained premenopausal. RESULTS: After a median follow-up of 67 months, the estimated disease-free survival rate at 5 years was 86.6% in the tamoxifen-ovarian suppression group and 84.7% in the tamoxifen group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.83; 95% confidence interval [CI], 0.66 to 1.04; P=0.10). Multivariable allowance for prognostic factors suggested a greater treatment effect with tamoxifen plus ovarian suppression than with tamoxifen alone (hazard ratio, 0.78; 95% CI, 0.62 to 0.98). Most recurrences occurred in patients who had received prior chemotherapy, among whom the rate of freedom from breast cancer at 5 years was 82.5% in the tamoxifen-ovarian suppression group and 78.0% in the tamoxifen group (hazard ratio for recurrence, 0.78; 95% CI, 0.60 to 1.02). At 5 years, the rate of freedom from breast cancer was 85.7% in the exemestane-ovarian suppression group (hazard ratio for recurrence vs. tamoxifen, 0.65; 95% CI, 0.49 to 0.87). CONCLUSIONS: Adding ovarian suppression to tamoxifen did not provide a significant benefit in the overall study population. However, for women who were at sufficient risk for recurrence to warrant adjuvant chemotherapy and who remained premenopausal, the addition of ovarian suppression improved disease outcomes. Further improvement was seen with the use of exemestane plus ovarian suppression. (Funded by Pfizer and others; SOFT ClinicalTrials.gov number, NCT00066690.).

BACKGROUND: Tranexamic acid reduces the risk of bleeding among patients undergoing cardiac surgery, but it is unclear whether this leads to improved outcomes. Furthermore, there are concerns that tranexamic acid may have prothrombotic and proconvulsant effects. METHODS: In a trial with a 2-by-2 factorial design, we randomly assigned patients who were scheduled to undergo coronary-artery surgery and were at risk for perioperative complications to receive aspirin or placebo and tranexamic acid or placebo. The results of the tranexamic acid comparison are reported here. The primary outcome was a composite of death and thrombotic complications (nonfatal myocardial infarction, stroke, pulmonary embolism, renal failure, or bowel infarction) within 30 days after surgery. RESULTS: Of the 4662 patients who were enrolled and provided consent, 4631 underwent surgery and had available outcomes data; 2311 were assigned to the tranexamic acid group and 2320 to the placebo group. A primary outcome event occurred in 386 patients (16.7%) in the tranexamic acid group and in 420 patients (18.1%) in the placebo group (relative risk, 0.92; 95% confidence interval, 0.81 to 1.05; P=0.22). The total number of units of blood products that were transfused during hospitalization was 4331 in the tranexamic acid group and 7994 in the placebo group (P<0.001). Major hemorrhage or cardiac tamponade leading to reoperation occurred in 1.4% of the patients in the tranexamic acid group and in 2.8% of the patients in the placebo group (P=0.001), and seizures occurred in 0.7% and 0.1%, respectively (P=0.002 by Fisher's exact test). CONCLUSIONS: Among patients undergoing coronary-artery surgery, tranexamic acid was associated with a lower risk of bleeding than was placebo, without a higher risk of death or thrombotic complications within 30 days after surgery. Tranexamic acid was associated with a higher risk of postoperative seizures. (Funded by the Australian National Health and Medical Research Council and others; ATACAS Australia New Zealand Clinical Trials Registry number, ACTRN12605000557639 .).

BACKGROUND: Directional coronary atherectomy is a new technique of coronary revascularization by which atherosclerotic plaque is excised and retrieved from target lesions. With respect to the rate of restenosis and clinical outcomes, it is not known how this procedure compares with balloon angioplasty, which relies on dilation of the plaque and vessel wall. We compared the rate of restenosis after angioplasty with that after atherectomy. METHODS: At 35 sites in the United States and Europe, 1012 patients were randomly assigned to either atherectomy (512 patients) or angioplasty (500 patients). The patients underwent coronary angiography at base line and again after six months; the paired angiograms were quantitatively assessed at one laboratory by investigators unaware of the treatment assignments. RESULTS: Stenosis was reduced to 50 percent or less more often with atherectomy than with angioplasty (89 percent vs. 80 percent; P < 0.001), and there was a greater immediate increase in vessel caliber (1.05 vs. 0.86 mm, P < 0.001). This was accompanied by a higher rate of early complications (11 percent vs. 5 percent, P < 0.001) and higher in-hospital costs ($11,904 vs $10,637; P = 0.006). At six months, the rate of restenosis was 50 percent for atherectomy and 57 percent for angioplasty (P = 0.06). However, the probability of death or myocardial infarction within six months was higher in the atherectomy group (8.6 percent vs. 4.6 percent, P = 0.007). CONCLUSIONS: Removing coronary artery plaque with atherectomy led to a larger luminal diameter and a small reduction in angiographic restenosis, the latter being confined largely to the proximal left anterior descending coronary artery. However, atherectomy led to a higher rate of early complications, increased cost, and no apparent clinical benefit after six months of follow-up.

BACKGROUND: Hypophosphatasia results from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates extracellularly, leading to rickets or osteomalacia. Severely affected babies often die from respiratory insufficiency due to progressive chest deformity or have persistent bone disease. There is no approved medical therapy. ENB-0040 is a bone-targeted, recombinant human TNSALP that prevents the manifestations of hypophosphatasia in Tnsalp knockout mice. METHODS: We enrolled infants and young children with life-threatening or debilitating perinatal or infantile hypophosphatasia in a multinational, open-label study of treatment with ENB-0040. The primary objective was the healing of rickets, as assessed by means of radiographic scales. Motor and cognitive development, respiratory function, and safety were evaluated, as well as the pharmacokinetics and pharmacodynamics of ENB-0040. RESULTS: Of the 11 patients recruited, 10 completed 6 months of therapy; 9 completed 1 year. Healing of rickets at 6 months in 9 patients was accompanied by improvement in developmental milestones and pulmonary function. Elevated plasma levels of the TNSALP substrates inorganic pyrophosphate and pyridoxal 5'-phosphate diminished. Increases in serum parathyroid hormone accompanied skeletal healing, often necessitating dietary calcium supplementation. There was no evidence of hypocalcemia, ectopic calcification, or definite drug-related serious adverse events. Low titers of anti-ENB-0040 antibodies developed in four patients, with no evident clinical, biochemical, or autoimmune abnormalities at 48 weeks of treatment. CONCLUSIONS: ENB-0040, an enzyme-replacement therapy, was associated with improved findings on skeletal radiographs and improved pulmonary and physical function in infants and young children with life-threatening hypophosphatasia. (Funded by Enobia Pharma and Shriners Hospitals for Children; ClinicalTrials.gov number, NCT00744042.).

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases. Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases. AMP-activated protein kinase endothelial nitric-oxide synthase bovine aortic endothelial cells human umbilical vein endothelial cells vascular endothelial growth factor isobutyl methylxanthine phospholipase C and D cAMP-dependent protein kinase protein kinase C Ca2+-calmodulin phorbol 12-myristate 13-acetate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein kinases involved in the regulation of endothelial NO production and eNOS activity include AMPK,1 PKA, PKB/Akt, PKC, and the calmodulin-dependent kinase II. Initially AMPK was shown to mediate ischemia-induced activation of eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar), but multiple stimuli including vascular endothelial growth factor (VEGF) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar), insulin-like growth factor-1 (IGF-1) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), estrogen (4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar), and fluid shear stress (6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) signal through Akt/PKB kinase to activate eNOS by Ser-1177 phosphorylation. Other vasoactive substances that elevate intracellular calcium (Ca2+) also regulate eNOS activity through Ca2+-calmodulin (CaM) binding (8Bernier S.G. Haldar S. Michel T. J. Biol. Chem. 2000; 275: 30707-30715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition to activating Akt/PKB, VEGF also activates PKC in endothelial cells (9Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (523) Google Scholar). Activation of both PLC and PLD by VEGF is accompanied by an early influx of Ca2+, which is inhibited by reduced extracellular Ca2+, PKC inhibitors, and tyrosine kinase inhibitors (10Seymour L.W. Shoaibi M.A. Martin A. Ahmed A. Elvin P. Kerr D.J. Wakelam M.J. Lab. Invest. 1996; 75: 427-437PubMed Google Scholar). Previously we found phosphorylation of Thr-495 by AMPK in vitro attenuated eNOS activity (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar) and recently reported that bradykinin activates eNOS in endothelial cells by triggering dephosphorylation at this site (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). Endothelial cell NOS activity is inhibited following phorbol 12,13-dibutyrate treatment (12Hirata K. Kuroda R. Sakoda T. Katayama M. Inoue N. Suematsu M. Kawashima S. Yokoyama M. Hypertension. 1995; 25: 180-185Crossref PubMed Google Scholar, 13Davda R.K. Chandler L.J. Guzman N.J. Eur. J. Pharmacol. 1994; 266: 237-244Crossref PubMed Scopus (47) Google Scholar). In the present study we show PKC signaling causes eNOS phosphorylation at Thr-495 as well as promoting dephosphorylation of Ser-1177. In contrast, PKA signaling results in phosphorylation of Ser-1177 and dephosphorylation of Thr-495 in endothelial cells. The dephosphorylation events are catalyzed by phosphatases PP1 and PP2A acting selectively on these two sites.DISCUSSIONThe regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently coordinated.Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 μm).VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. Protein kinases involved in the regulation of endothelial NO production and eNOS activity include AMPK,1 PKA, PKB/Akt, PKC, and the calmodulin-dependent kinase II. Initially AMPK was shown to mediate ischemia-induced activation of eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar), but multiple stimuli including vascular endothelial growth factor (VEGF) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar), insulin-like growth factor-1 (IGF-1) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), estrogen (4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar), and fluid shear stress (6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) signal through Akt/PKB kinase to activate eNOS by Ser-1177 phosphorylation. Other vasoactive substances that elevate intracellular calcium (Ca2+) also regulate eNOS activity through Ca2+-calmodulin (CaM) binding (8Bernier S.G. Haldar S. Michel T. J. Biol. Chem. 2000; 275: 30707-30715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition to activating Akt/PKB, VEGF also activates PKC in endothelial cells (9Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (523) Google Scholar). Activation of both PLC and PLD by VEGF is accompanied by an early influx of Ca2+, which is inhibited by reduced extracellular Ca2+, PKC inhibitors, and tyrosine kinase inhibitors (10Seymour L.W. Shoaibi M.A. Martin A. Ahmed A. Elvin P. Kerr D.J. Wakelam M.J. Lab. Invest. 1996; 75: 427-437PubMed Google Scholar). Previously we found phosphorylation of Thr-495 by AMPK in vitro attenuated eNOS activity (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar) and recently reported that bradykinin activates eNOS in endothelial cells by triggering dephosphorylation at this site (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). Endothelial cell NOS activity is inhibited following phorbol 12,13-dibutyrate treatment (12Hirata K. Kuroda R. Sakoda T. Katayama M. Inoue N. Suematsu M. Kawashima S. Yokoyama M. Hypertension. 1995; 25: 180-185Crossref PubMed Google Scholar, 13Davda R.K. Chandler L.J. Guzman N.J. Eur. J. Pharmacol. 1994; 266: 237-244Crossref PubMed Scopus (47) Google Scholar). In the present study we show PKC signaling causes eNOS phosphorylation at Thr-495 as well as promoting dephosphorylation of Ser-1177. In contrast, PKA signaling results in phosphorylation of Ser-1177 and dephosphorylation of Thr-495 in endothelial cells. The dephosphorylation events are catalyzed by phosphatases PP1 and PP2A acting selectively on these two regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently coordinated.Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 μm).VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. The regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google Scholar). The inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. We S. Shenolikar at for on okadaic acid and calyculin A.

BACKGROUND: Daratumumab, a monoclonal antibody targeting CD38, has been approved for use with standard myeloma regimens. An evaluation of subcutaneous daratumumab combined with bortezomib, lenalidomide, and dexamethasone (VRd) for the treatment of transplantation-eligible patients with newly diagnosed multiple myeloma is needed. METHODS: In this phase 3 trial, we randomly assigned 709 transplantation-eligible patients with newly diagnosed multiple myeloma to receive either subcutaneous daratumumab combined with VRd induction and consolidation therapy and with lenalidomide maintenance therapy (D-VRd group) or VRd induction and consolidation therapy and lenalidomide maintenance therapy alone (VRd group). The primary end point was progression-free survival. Key secondary end points were a complete response or better and minimal residual disease (MRD)-negative status. RESULTS: At a median follow-up of 47.5 months, the risk of disease progression or death in the D-VRd group was lower than the risk in the VRd group. The estimated percentage of patients with progression-free survival at 48 months was 84.3% in the D-VRd group and 67.7% in the VRd group (hazard ratio for disease progression or death, 0.42; 95% confidence interval, 0.30 to 0.59; P<0.001); the P value crossed the prespecified stopping boundary (P = 0.0126). The percentage of patients with a complete response or better was higher in the D-VRd group than in the VRd group (87.9% vs. 70.1%, P<0.001), as was the percentage of patients with MRD-negative status (75.2% vs. 47.5%, P<0.001). Death occurred in 34 patients in the D-VRd group and 44 patients in the VRd group. Grade 3 or 4 adverse events occurred in most patients in both groups; the most common were neutropenia (62.1% with D-VRd and 51.0% with VRd) and thrombocytopenia (29.1% and 17.3%, respectively). Serious adverse events occurred in 57.0% of the patients in the D-VRd group and 49.3% of those in the VRd group. CONCLUSIONS: The addition of subcutaneous daratumumab to VRd induction and consolidation therapy and to lenalidomide maintenance therapy conferred a significant benefit with respect to progression-free survival among transplantation-eligible patients with newly diagnosed multiple myeloma. (Funded by the European Myeloma Network in collaboration with Janssen Research and Development; PERSEUS ClinicalTrials.gov number, NCT03710603; EudraCT number, 2018-002992-16.).

We have examined the mechanisms underlying reduced circulating GH concentrations in the obese human. Computer-assisted (deconvolution) analysis was used to determine endogenous GH secretory and clearance rates quantitatively from entire 24-h plasma GH concentration profiles. These analyses revealed that the half-life (t 1/2) of endogenous GH was significantly shorter in obese (11.7 +/- 1.6 min) than in normal weight subjects (15.5 +/- 0.81 min; P less than 0.01). The accelerated blood disposal rate of GH was not due to decreased circulating concentrations of GH-binding protein, since the latter were similar in obese (25 +/- 1.0%) and normal weight (24 +/- 2.3%) men. However, obese men had significantly fewer GH secretory bursts (3.2 +/- 0.53 vs. 9.7 +/- 0.67/day; P less than 0.01). Among the rare GH secretory bursts that occurred in obese subjects, there were significantly prolonged mean intersecretory burst intervals (282 +/- 65 vs. 131 +/- 11 min; P less than 0.05). The resultant daily GH production rate in obese men was reduced to one fourth that in normal weight individuals. Both GH secretion rate and burst frequency were negatively correlated with the degree of obesity (ponderal index). The decreases in GH burst frequency and half-life were specific, since GH secretory pulse amplitude (maximal rate of GH release), the mass of GH released per burst, and the duration of computer-resolved GH secretory bursts were not different in obese and normal weight men. We conclude that obese men harbor a double defect in GH dynamics involving both GH secretion and clearance, and that the severity of the GH secretory deficiency is proportionate to the degree of obesity.

PURPOSE: To examine prevalence of refractive errors and its associated factors, such as body stature and educational level, among 19-year-old males in Seoul, Korea. METHODS: A population-based cross-sectional study was performed in male subjects (n = 23,616; age = 19 years) who were normally resident in Seoul for male compulsory conscripts during the study period (2010). Refractive examination was performed with cycloplegia. Height, weight, and educational level were examined. Myopia was defined as a spherical equivalent less than -0.5 diopters (D) and high myopia less than -6.0 D. The association of myopia with body stature and educational level was analyzed using logistic regression analysis. RESULTS: The prevalence of myopia in 19-year-old males in Seoul was 96.5%. The prevalence of high myopia was 21.61%. Body stature was not significantly associated with myopia. Four- to 6-year university students (odds ratio [OR] 1.69; P < 0.001) and 2 to 3-year college students (OR 1.68; P < 0.001) showed significantly higher risk for myopia than those with lower academic achievement (< high school graduation). CONCLUSIONS: The 19-year-old male population in Seoul, Korea, demonstrated a very high myopic prevalence. Myopic refractive error was associated with academic achievement, not with body stature.

BACKGROUND: Elevated serum urate levels are associated with progression of chronic kidney disease. Whether urate-lowering treatment with allopurinol can attenuate the decline of the estimated glomerular filtration rate (eGFR) in patients with chronic kidney disease who are at risk for progression is not known. METHODS: of body-surface area in the preceding year to receive allopurinol (100 to 300 mg daily) or placebo. The primary outcome was the change in eGFR from randomization to week 104, calculated with the Chronic Kidney Disease Epidemiology Collaboration creatinine equation. RESULTS: per year [95% CI, -1.18 to 0.97]; P = 0.85). Serious adverse events were reported in 84 of 182 patients (46%) in the allopurinol group and in 79 of 181 patients (44%) in the placebo group. CONCLUSIONS: In patients with chronic kidney disease and a high risk of progression, urate-lowering treatment with allopurinol did not slow the decline in eGFR as compared with placebo. (Funded by the National Health and Medical Research Council of Australia and the Health Research Council of New Zealand; CKD-FIX Australian New Zealand Clinical Trials Registry number, ACTRN12611000791932.).