Post-transplant lymphoproliferative disorders (PTLD)-from clinical to metabolic profiles-a single center experience and review of literature.

Post-transplant lymphoproliferative disorders (PTLD) are among the most serious complications after solid organ transplantation (SOT). Monomorphic diffuse large B-cell lymphoma (DLBCL) is the most common subtype of PTLD. Historically, outcomes of PTLD have been poor with high mortality rates and allograft loss, although this has improved in the last 10 years. Most of our understanding about PTLD DLBCL is extrapolated from studies in non-PTLD DLBCL, and while several clinical factors have been identified and validated for predicting non-PTLD DLBCL outcomes, the molecular profile of PTLD DLBCL has not yet been characterized. Compartment-specific metabolic reprograming has been described in non-PTLD DLBCL with a lactate uptake metabolic phenotype with high monocarboxylate transporter 1 (MCT1) expression associated with worse outcomes. The aim of our study was to compare the outcomes of PTLD in our transplant center to historic cohorts, as well as study a subgroup of our PTLD DLBCL tumors and compare metabolic profiles with non-PTLD DLBCL. We performed a retrospective single institution study of all adult patients who underwent a SOT between the years 1992-2018, who were later diagnosed with PTLD. All available clinical information was extracted from the patients' medical records. Tumor metabolic markers were studied in a subgroup of PTLD DLBCL and compared to a group of non-PTLD DLBCL. Thirty patients were diagnosed with PTLD following SOT in our center. Median time from SOT to PTLD diagnosis was 62.8 months (IQR 7.6; 134.4), with 37% of patients diagnosed with early PTLD, and 63% with late PTLD. The most common PTLD subtype was DLBCL. Most patients were treated with reduction of their immunosuppression (RIS) including a group who were switched from calcineurin inhibitor (CNI) to mTOR inhibitor based IS, in conjunction with standard anti-lymphoma chemoimmunotherapy. Progression free survival of the PTLD DLBCL cohort was calculated at 86% at 1 year, and 77% at 3 and 5-years, with overall survival of 86% at 1 and 3-years, and 75% at 5 years. Death censored allograft survival in the kidney cohort was 100% at 1 year, and 93% at 3, 5 and 10 years. MCT1 H scores were significantly higher in a subset of the non-PTLD DLBCL patients than in a PTLD DLBCL cohort. Our data is concordant with improved PTLD outcomes in the last 10 years. mTOR inhibitors could be an alternative to CNI as a RIS strategy. Finally, PTLD DLBCL may have a distinct metabolic profile with reduced MCT1 expression compared to non-PTLD DLBCL, but further studies are needed to corroborate our limited cohort findings and to determine if a specific metabolic profile is associated with outcomes.

Macrophage and adipocyte interaction as a source of inflammation in kidney disease.

In obesity, adipose tissue derived inflammation is associated with unfavorable metabolic consequences. Uremic inflammation is prevalent and contributes to detrimental outcomes. However, the contribution of adipose tissue inflammation in uremia has not been characterized. We studied the contribution of adipose tissue to uremic inflammation in-vitro, in-vivo and in human samples. Exposure to uremic serum resulted in activation of inflammatory pathways including NFκB and HIF1, upregulation of inflammatory cytokines/chemokines and catabolism with lipolysis, and lactate production. Also, co-culture of adipocytes with macrophages primed by uremic serum resulted in higher inflammatory cytokine expression than adipocytes exposed only to uremic serum. Adipose tissue of end stage renal disease subjects revealed increased macrophage infiltration compared to controls after BMI stratification. Similarly, mice with kidney disease recapitulated the inflammatory state observed in uremic patients and additionally demonstrated increased peripheral monocytes and inflammatory polarization of adipose tissue macrophages (ATMS). In contrast, adipose tissue in uremic IL-6 knock out mice showed reduced ATMS density compared to uremic wild-type controls. Differences in ATMS density highlight the necessary role of IL-6 in macrophage infiltration in uremia. Uremia promotes changes in adipocytes and macrophages enhancing production of inflammatory cytokines. We demonstrate an interaction between uremic activated macrophages and adipose tissue that augments inflammation in uremia.

Diabetes in Kidney Transplantation.

Diabetes mellitus (DM) is one of the most common complications after kidney transplantation and is associated with unfavorable outcomes including death. DM can be present before transplant but post-transplant DM (PTDM) refers to diabetes that is diagnosed after solid organ transplantation. Despite its high prevalence, optimal treatment to prevent complications of PTDM is unknown. Medical therapy of pre-existent DM or PTDM after transplant is challenging because of frequent interactions between antidiabetic and immunosuppressive agents. There is also frequent need for medication dose adjustments due to residual kidney disease and a higher risk of medication side effects in patients treated with immunosuppressive agents. Sodium-glucose cotransporter 2 inhibitors have demonstrated a favorable cardio-renal profile in patients with DM without a transplant and hence hold great promise in this patient population although there is concern about the higher risk of urinary tract infections. The significant gaps in our understanding of the pathophysiology, diagnosis, and management of DM after kidney transplantation need to be urgently addressed.

Correction to: Effect of CYP3A Inhibition and Induction on the Pharmacokinetics of Suvorexant: Two Phase I, Open-Label, Fixed-Sequence Trials in Healthy Subjects.

The original version of this article unfortunately contained a mistake.

Effect of CYP3A Inhibition and Induction on the Pharmacokinetics of Suvorexant: Two Phase I, Open-Label, Fixed-Sequence Trials in Healthy Subjects.

BACKGROUND AND OBJECTIVES: Suvorexant is an orexin receptor antagonist indicated for the treatment of insomnia, characterized by difficulties with sleep onset and/or sleep maintenance. As suvorexant is metabolized primarily by Cytochrome P450 3A (CYP3A), and its pharmacokinetics may be affected by CYP3A modulators, the effects of CYP3A inhibitors (ketoconazole or diltiazem) or an inducer (rifampin [rifampicin]) on the pharmacokinetics, safety, and tolerability of suvorexant were investigated. METHODS: In two Phase I, open-label, fixed-sequence trials (Studies P008 and P038), healthy subjects received a single oral dose of suvorexant followed by co-administration with multiple once-daily doses of strong/moderate CYP3A inhibitors (ketoconazole/diltiazem) or a strong CYP3A inducer (rifampin). Treatments were administered in the morning: suvorexant 4 mg with ketoconazole 400 mg (Study P008; N = 10), suvorexant 20 mg with diltiazem 240 mg (Study P038; N = 20), and suvorexant 40 mg with rifampin 600 mg (Study P038; N = 10). Area under the plasma concentration-time curve from time zero to infinity (AUC0-∞), maximum plasma concentration (Cmax), half-life (t½), and time to Cmax (tmax) were derived from plasma concentrations of suvorexant collected at prespecified time points up to 10 days following CYP3A inhibitor/inducer co-administration. Adverse events (AEs) were recorded. RESULTS: Co-administration with ketoconazole resulted in increased exposure to suvorexant [AUC0-∞: geometric mean ratio (GMR); 90% confidence interval (CI) 2.79 (2.35, 3.31)] while co-administration with diltiazem resulted in a lesser effect [GMR (90% CI): 2.05 (1.82, 2.30)]. Co-administration with rifampin led to a marked decrease (88%) in suvorexant exposure. Consistent with morning administration and known suvorexant pharmacology, somnolence was the most frequently reported AE. CONCLUSIONS: These results are consistent with expectations that strong CYP3A inhibitors and inducers exert marked effects on suvorexant pharmacokinetics. In the context of a limited sample size, single suvorexant doses were generally well tolerated in healthy subjects when co-administered with/without a CYP3A inhibitor/inducer.

Uremia induces adipose tissue inflammation and muscle mitochondrial dysfunction.

BACKGROUND.: End-stage renal disease (ESRD) is associated with inflammation and increased reactive oxygen species (ROS). Inflammation and oxidative stress are associated with several complications of ESRD. The aim of this study was to determine histological characteristics of adipose tissue and muscle mitochondrial function in uremia and its relationship with inflammation. METHODS.: ESRD patients ( n = 18) and controls ( n = 6) were enrolled for studies of adipose and muscle tissue by immunohistochemistry and western blot. In a uremic muscle cell model, C2C12 cells were exposed to uremic serum and inflammatory cytokines. Mitochondrial function was studied by MitoTracker Orange, translocase of the mitochondrial outer membrane 20 (TOMM20) and mitochondrial oxidative phosphorylation complex subunit expression. RESULTS.: ESRD patients had increased macrophage infiltration in subcutaneous and visceral adipose tissue compared with controls, even in nonobese ESRD patients (P < 0.05). Compared with controls, TOMM20 expression in muscle tissue was lower in ESRD, consistent with reduced mitochondrial function (P < 0.05). C2C12 exposed to uremia had decreased mitotracker intensity (P < 0.05) and the reduced mitochondrial function was rescued by N-acetyl cysteine (P < 0.01). Similarly, C2C12 cells exposed to tumor necrosis factor α (TNF-α)/interleukin-6 (IL-6) have decreased mitotracker intensity (P < 0.01) that was rescued with adiponectin (P < 0.05). C2C12 exposed to TNF-α, IL-6 and buthionine sulfoximine had decreased TOMM20 expression and cells exposed to TNF-α showed a decrease in subunits of mitochondrial complexes I and III. CONCLUSION.: Our data indicate that uremia is associated with increased adipose tissue macrophage infiltration and concurrent muscle tissue mitochondrial dysfunction induced by inflammation/ROS. Adipose tissue is a potential source of inflammation in ESRD that is not due to increased adiposity and may contribute to mitochondrial dysfunction in uremia.

Correlation of acute tubular injury in reperfusion biopsy with renal transplant outcomes.

Acute tubular injury (ATI) is common at reperfusion, but its relationship to graft outcomes is unclear. Prior studies lack standardization of morphological assessments and included elements of acute and chronic tubular injury. This study aimed to evaluate the impact of ATI on graft outcomes. Reperfusion biopsies from 2004 to 2009 were retrospectively reviewed. ATI was assessed by a new standardized scoring system. We also assessed chronic injury (CI) by the Banff criteria. Outcomes evaluated included glomerular filtration rate (GFR) at 1 and 5 years and delayed graft function (DGF), acute rejection (AR), graft and patient survival. ATI did not correlate with DGF, AR, graft or overall survival. Mild-moderate ATI was not predictive of GFR post-transplant. Moderate-severe CI was associated with lower GFR at 5 years with a mean difference of -7.14 mL/min/1.73 m(2) (P=.04) and overall survival (HR 2.44, P=.01). Other predictors of graft function included donor age, DGF, and AR. Histologic criteria of ATI at implantation in the absence of donor demographics or clinical information do not provide sufficient predictability in outcomes after transplantation. On the other hand, histologic assessment of CI correlates with GFR and overall survival.

Parathyroid Hormone-Related Peptide-Linked Hypercalcemia in a Melanoma Patient Treated With Ipilimumab: Hormone Source and Clinical and Metabolic Correlates.

A patient diagnosed with metastatic melanoma developed the paraneoplastic syndrome of humoral hypercalcemia of malignancy and cachexia after receiving ipilumumab. The cause of the hypercalcemia was thought to be secondary to parathyroid hormone-related peptide (PTHrP) as plasma levels were found to be elevated. The patient underwent two tumor biopsies: at diagnosis (when calcium levels were normal) and upon development of hypercalcemia and cachexia. PTHrP expression was higher in melanoma cells when hypercalcemia had occurred than prior to its onset. Metabolic characterization of melanoma cells revealed that, with development of hypercalcemia, there was high expression of monocarboxylate transporter 1 (MCT1), which is the main importer of lactate and ketone bodies into cells. MCT1 is associated with high mitochondrial metabolism. Beta-galactosidase (ß-GAL), a marker of senescence, had reduced expression in melanoma cells upon development of hypercalcemia compared to pre-hypercalcemia. In conclusion, PTHrP expression in melanoma is associated with cachexia, increased cancer cell lactate and ketone body import, high mitochondrial metabolism, and reduced senescence. Further studies are required to determine if PTHrP regulates cachexia, lactate and ketone body import, mitochondrial metabolism, and senescence in cancer cells.

Adiponectin receptor and adiponectin signaling in human tissue among patients with end-stage renal disease.

BACKGROUND: Adiponectin plasma levels in chronic kidney disease (CKD) are two to three times higher than in individuals with normal kidney function. Despite adiponectin's anti-diabetic, anti-inflammatory and anti-atherogenic properties, patients with CKD have insulin resistance, systemic inflammation and accelerated atherogenesis. Hence, although adiponectin production is increased by adipose tissue in end-stage renal disease (ESRD), it is unclear if its effects on metabolism remain intact. METHODS: To determine if there is adiponectin resistance in ESRD, we measured tissue levels of adiponectin receptor-1 (AdipoR1) and adiponectin downstream effectors in ESRD patients compared with normal kidney function controls. Blood and tissue samples were obtained from participants at the time of kidney transplantation or kidney donation. A follow-up blood sample was obtained 3-6 months after transplantation. RESULTS: AdipoR1 was higher in muscle and peripheral blood mononuclear cells collected from ESRD patients. There was also a nonsignificant increase in AdipoR1 in visceral fat of ESRD compared with controls. Compared with controls, phosphorylation of the adiponectin downstream effector adenosine monophosphate-activated protein kinase (AMPK) was higher in ESRD while acetyl-CoA carboxylase phosphorylation (ACC-P) and carnitine palmitoyl transferase-1 (CPT-1) levels were lower. In vitro, exposure of C2C12 cells to uremic serum resulted in upregulation of AdipoR1 and increased phosphorylation of AMPK but decreased ACC-P and CPT-1 expression. CONCLUSION: Both our in vivo and in vitro observations indicate that uremia results in upregulation of AdipoR1 but adiponectin resistance at the post-receptor level.

The adipose tissue production of adiponectin is increased in end-stage renal disease.

Adiponectin has antidiabetic properties, and patients with obesity, diabetes, and insulin resistance have low plasma adiponectin levels. However, although kidney disease is associated with insulin resistance, adiponectin is elevated in end-stage renal disease. Here we determine whether adipose tissue production of adiponectin is increased in renal disease in a case-control study of 36 patients with end-stage renal disease and 23 kidney donors. Blood and tissue samples were obtained at kidney transplantation and donation. The mean plasma adiponectin level was significantly increased to 15.6 mg/ml in cases compared with 8.4 mg/ml in controls. Plasma levels of the inflammatory adipokines tumor necrosis factor α, interleukin 6, and high-sensitivity C-reactive protein were significantly higher in cases compared with controls. Adiponectin mRNA and protein expression in visceral and subcutaneous fat were significantly higher in cases than controls, while adiponectin receptor-1 mRNA expression was significantly increased in peripheral blood cells, muscle, and adipose tissue in cases compared with controls. Thus, our study suggests that adipose tissue production of adiponectin contributes to the high plasma levels seen in end-stage renal disease.

Relationship of blood pressure and obesity with inflammatory cytokines among African Americans.

OBJECTIVES: Hypertension and obesity are major public health issues. Both conditions are highly prevalent among African Americans and contribute to the increased burden of cardiovascular disease in this group. Inflammation is considered to be an underlying process in both conditions. The authors sought to determine if there is an interaction between high blood pressure (HBP) and obesity that is associated with markedly elevated plasma levels of proinflammatory cytokines in African American adults. METHODS: This study examined 484 African Americans, aged 18-45 years, including people with and without obesity, and also with and without HBP. People known to have diabetes were not enrolled. Plasma levels of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), plasminogen activator inhibitor 1, tumor necrosis factor alpha (TNF-α), and adiponectin were measured. Participants also underwent an oral glucose tolerance test and measurement of blood pressure and body mass index (BMI). RESULTS: There was no statistically significant interaction between obesity and HBP on plasma levels of adiponectin or the inflammatory cytokines. When both conditions were present, HBP and obesity had additive associations with the expected geometric mean ratios for IL-6 (1.44, 95% CI 1.18 to 1.75), TNF-α (1.31, 95% CI 1.11 to 1.53), hsCRP (2.55, 95% CI 1.99 to 3.26) and negative association with adiponectin (0.61, 95% CI 0.52 to 0.71). Compared with HBP, obesity had the predominant association with cytokine levels. An increase in cytokine plasma levels was detectable when BMI exceeded 25 kg/m2. CONCLUSIONS: Biomarkers of inflammation in African Americans are strongly associated with BMI. A modest additive effect is found with HBP. Interventions to reduce obesity-related inflammation may impact cardiovascular disease outcomes.

Relationship of adipokines with insulin sensitivity in African Americans.

INTRODUCTION: Cytokines produced by adipose tissue, including adiponectin, have been associated with metabolic abnormalities. The purpose of this study was to examine the relationship of insulin sensitivity measured by euglycemic hyperinsulinemic insulin clamp with plasma adiponectin and other adipokines in young adult African Americans. METHODS: Participants were healthy African Americans. Anthropometric measures, blood pressure, an oral glucose tolerance test and an euglycemic hyperinsulinemic insulin clamp were performed. Insulin sensitivity measurements were adjusted for percentage of fat mass. Plasma concentrations of adiponectin, plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6) were assayed on plasma from fasting blood samples. Pearson correlation coefficients and multiple regression models were fitted to assess the association between glucose sensitivity and cytokines. RESULTS: In univariate analysis, there were statistically significant correlations of plasma adiponectin level (r = 0.19, P = 0.004), PAI-1 (r = -0.19, P = 0.020) and IL-6 (r = -0.24, P < 0.001) with measures of insulin sensitivity after adjustment for both fat mass and insulin clamp concentration. In multivariate analysis, adiponectin [geometric mean ratios (GMR) 1.15, P = 0.007], PAI-1 (GMR 0.998, P = 0.021) and body mass index (GMR 0.95, P < 0.001) were each independently associated with insulin sensitivity. For IL-6, there was no significant association with insulin sensitivity independent of obesity. CONCLUSION: These data show a significant and independent positive correlation of adiponectin with insulin sensitivity. The relationship of IL-6 with insulin sensitivity seems to be dependent on adiposity.

Variants in genes involved in functional pathways associated with hypertension in African Americans.

Essential hypertension (HBP) is a complex trait with a substantial heritable component. The purpose of this study was to determine if variants in the G-protein coupled receptor Kinase-4 (GRK4), nitric oxide synthase-3 (NOS3), or angiotensin converting enzyme (ACE) genes are associated singly or through complex interactions, with HBP in African Americans aged 18-49 years. TaqMan Assays were used for genotyping the GRK4 and NOS3 variants. The ACE I/D variant was obtained by polymerase chain reaction and electrophoresis. Allelic association tests were performed for the five markers using PLINK. Logistic regression models were fitted to investigate associations between HBP status and the genetic markers. Multilocus analyses were also conducted. The study included 173 hypertensives and 239 normotensives, with stratification into obese and nonobese groups. The GRK4 A486V variant was negatively associated with HBP in the nonobese group (p = 0.048). The TT/CT genotype of GRK4 A486V was associated with decreased risk for HBP relative to the CC genotype after adjusting for age, sex, and body mass index (p = 0.028). Individuals having at least one NOS3 A allele and GRK4 R65L genotype GG had odds of HBP of 2.97 relative to GG homozygotes for NOS3 and GRK4 R65L. These results show very modest effects and do not fully replicate previous studies.

Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NFκB activation in the tumor stromal microenvironment.

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB--another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism", and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFß/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].