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Background: Extracellular renal interstitial guanosine cyclic 3’,5’-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na + ) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na + transport similar to ouabain—a cardiotonic steroid. Methods: Urine Na + excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA. Results: Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated Src Tyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2) Thr202/Tyr204 ; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N 3 -PET-8-biotin-11-cGMP (2 μM); 8-N 3 -6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N 3 -PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non–cross-linked samples and cross-linked or non–cross-linked 8-N 3 -6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N 3 -PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N 3 -PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. Conclusions: cGMP can bind to NKA and thereby mediate natriuresis.

Movement of cells in anti-CD49b treated tumor slices

CD8+ T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8+ T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8+ T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8+ T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8+ T-cell dysfunction.

PDF file, 340K, CDT1 levels in SKOV3 and ES2 cells.

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PDF file, 400K, MLN4924 and cisplatin enhance apoptosis induction in ovarian cancer cells.

Purpose: Minimally invasive methods of predicting the risk of muscle-invasive urothelial bladder carcinoma may expedite appropriate therapy and reduce morbidity and cost.Experimental Design: Here, capillary electrophoresis coupled mass spectrometry was used to identify urinary polypeptide bladder cancer biomarkers in 127 patients. These markers were used to construct a panel discriminating muscle-invasive from noninvasive disease, which was refined in 297 additional samples from healthy volunteers, patients with malignant and nonmalignant genitourinary conditions. Sequencing of panel polypeptides was then done. Finally, the ability of the panel to predict muscle-invasive disease was evaluated prospectively in 130 bladder carcinoma patients. Four sequenced polypeptides formed a panel predictive of muscle-invasive disease.Results: Prospective evaluation of this panel revealed a sensitivity of 81% [95% confidence interval (CI), 69-90] and specificity of 57% (95% CI, 45-69) for muscle-invasive disease. Multivariate analysis revealed the panel (P < 0.0001) and tumor grade (P = 0.0001), but not urine cytology, predict muscle invasion. A model including grade and panel polypeptide levels improved sensitivity [92% (95% CI, 82-97)] and specificity [68% (95% CI, 55-79)] for muscle-invasive disease. A model score of >0.88 provided a negative predictive value of 77% and positive predictive value of 90% for muscle invasion.Conclusions: Use of urinary peptides seems promising in estimating the probability a patient harbors muscle-invasive urothelial bladder cancer. These peptides may also shed novel insights into the biology of bladder tumor progression not obtainable by other methods. Clinical trials seem warranted to evaluate the effect of this approach on practice.

Supplemental Table S1. Parameters for generating toxicity, response and median survival profiles for a population of agents Supplementary Figure S1. 100 randomly selected drug profiles, A: toxicity; B: response rate; C: median survival for the population that matches agents entering the pipeline from 1993-2004 (section 2.6) Supplementary Figure S2. Plot of 100 randomly selected toxicity, reponse and median survival profiles for 4, 6 or 8 doses levels to be tested in the phase I trial (section 2.6) Supplemental Table S2. Proportion of drugs with two successful phase III trials among 17225 drugs with a phase I trial that has at least one safe dose, defined as a toxicity probability less than or equal to 20% (section 3.2) Supplemental Table S3.. Proportion of drugs with two successful phase III trials among 20000 drugs in the 'low toxicity' population (section 3.3) Supplemental Table S4. Proportion of drugs with two successful phase III trials using an initial phase I target of 30% (section 3.2) Supplementary Figure S4. Plot of proportion of agents with two successful phase II trials using a target of 30% for CRM and BOIN (section 3.2)

Supplementary Data from Prediction of Muscle-invasive Bladder Cancer Using Urinary Proteomics

Purpose:Many of the therapeutic agents that are being used currently were developed using the 3+3 decision rule for dose finding. Over the past 30 years, several dose-finding designs have been proposed and evaluated, including the “continual reassessment method” (CRM) and the “Bayesian optimal interval design” (BOIN). This research investigates the role of the choice of an early-phase design on the likelihood that drugs entering the drug development pipeline will have 2 successful phase III trials.Experimental Design: Using simulation, each agent in a population of hypothetical agents was tracked through the drug development process, from initial dose finding to 2 confirmatory phase III trials. Varying the designs of the phase I, II, and III trials allows for an assessment of the effect of the choice of designs on the proportion of agents with successful phase III trials.Results:The results indicate that using the CRM or BOIN, rather than the 3+3, substantially enhances the proportion of effective agents that have successful phase III trials, with the CRM having a greater effect than BOIN. A larger phase II trial magnifies the effect of the phase I design.Conclusions:The results underscore the importance of the choice of the early-phase designs. Use of the 3+3 results in fewer agents with successful phase III trials compared with the CRM or BOIN. The difference is more pronounced among highly effective agents. In addition, the results show the importance of a sufficiently powered phase II trial.

Supplementary Figure 2 from Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis

Primer Sequences.

Supplementary Figure Legends and Tables 1-2 from Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis

RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paralogue-specific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphosite-specific antibodies, here we show phosphorylation of RalB by protein kinase C (PKC) and RalA by protein kinase A. We used mass spectrometry and site-directed mutagenesis to identify S198 as the primary PKC phosphorylation site in RalB. Phorbol ester [phorbol 12-myristate 13-acetate (PMA)] treatment of human bladder carcinoma cells induced S198 phosphorylation of stably expressed FLAG-RalB as well as endogenous RalB. PMA treatment caused RalB translocation from the plasma membrane to perinuclear regions in a S198 phosphorylation–dependent manner. Using RNA interference depletion of RalB followed by rescue with wild-type RalB or RalB(S198A) as well as overexpression of wild-type RalB or RalB(S198A) with and without PMA stimulation, we show that phosphorylation of RalB at S198 is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis of T24 or UMUC3 human bladder cancer cells. In addition, UMUC3 cells transfected with a constitutively active RalB(G23V) exhibited enhanced subcutaneous tumor growth, whereas those transfected with phospho-deficient RalB(G23V-S198A) were indistinguishable from control cells. Our data show that RalA and RalB are phosphorylated by different kinases, and RalB phosphorylation is necessary for in vitro cellular functions and in vivo tumor growth and metastasis. Cancer Res; 70(21); 8760–9. ©2010 AACR.

Supplementary Figure 1 from Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis

This article addresses the problem of identifying the maximum tolerated dose (MTD) in Phase I dose-finding clinical trials with late-onset toxicities. The main design challenge is how best to adaptively allocate study participants to tolerable doses when the evaluation window for the toxicity endpoint is long relative to the accrual rate of new participants. We propose a new design framework based on order-restricted statistical inference that addresses this challenge in sequential dose assignments. We illustrate the proposed method on real data from a Phase I trial of bortezomib in lymphoma patients and apply it to a Phase I trial of radiotherapy in prostate cancer patients. We conduct extensive simulation studies to compare our design's operating characteristics to existing published methods. Overall, our proposed design demonstrates good performance relative to existing methods in allocating participants at and around the MTD during the study and accurately recommending the MTD at the study conclusion.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Truncus arteriosus (TA) is a major congenital cardiac malformation that requires surgical repair in the first few weeks of life. Interrupted aortic arch (IAA) is an associated malformation that significantly impacts the complexity of the TA operation. The aim of this study was to (1) define the comorbid conditions associated with TA and (2) determine the hospital survival and morbidity of patients with TA with and without an IAA. Data was collected from the Vizient Clinical Database/Resource Manager, formerly University HealthSystem Consortium, which encompasses more than 160 academic medical centers in the United States. The database was queried for patients admitted from 2002 to 2016 who were ≤ 4 months of age at initial admission, diagnosed with TA, and underwent complete surgical repair during that hospitalization. Of the 645 patients with TA who underwent surgery, 98 (15%) had TA with an interrupted aortic arch (TA-IAA). Both TA and TA-IAA were associated with a high prevalence of comorbidities, including DiGeorge syndrome, prematurity, and other congenital malformations. There was no difference in mortality between TA and TA-IAA (13.7–18.4%, p value = 0.227). No comorbid conditions were associated with an increased mortality in either group. However, patients with TA-IAA had a longer post-operative length of stay (LOS) compared to those without IAA (30 versus 40.3 days, p value = 0.001) and this effect was additive with each additional comorbid condition. In conclusion, the addition of IAA to TA is associated with an increased post-operative LOS, but does not increase in-hospital mortality.

Objective Infants with a history of neonatal hypoxic-ischemic encephalopathy (HIE) are at risk for oral motor dysfunction. Previous studies have associated the need for gastrostomy tube at neonatal intensive care unit discharge with brainstem injury on magnetic resonance imaging (MRI). However, the factors associated with time to full oral feeds in this population have not been previously described. This study aimed to study factors associated with time to full oral feeds in this population. Study design This is a single-center, retrospective study that examined these factors using Cox regression. Results A total of 150 infants who received therapeutic hypothermia from 2011 to 2017 were included in this study. The single clinical factor significantly associated with time to full oral feeds was the severity of background abnormality on electroencephalogram in the first 24 hours of age (severe vs. mild 95% confidence interval [CI]: 0.34-0.74; moderate vs. mild 95% CI: 0.19-0.45). Brainstem injury on MRI was the factor most highly associated with need for gastrostomy tube placement (p = 0.028), though the overall incidence of need for gastrostomy tube feeds in this population was low (5%). Conclusion These findings may help clinicians counsel families on what to expect in neonates with HIE and make decisions on the need for and timing to pursue gastrostomy tube in this population. Key points · The overall incidence of the need for assisted feeding at NICU discharge is low in this population.. · MRI brainstem injury was most highly associated with need for gastrostomy tube placement.. · Worsening severity of background abnormality on EEG was associated with longer time to oral feeds..

Lactoferrin as a nutritional enteral supplement has emerged as a novel preventative therapy against serious infections in preterm infants, although neonatal studies have demonstrated variable results, in part due to the lack of pharmacokinetic data and differences in the products tested. We conducted a prospective, dose escalation (100, 200, and 300 mg·kg–1·day–1) safety study of bovine lactoferrin (Glanbia Nutritionals, USA) dissolved in sterile water (100 mg·mL–1) for 30 days in preterm infants with birth weight –1·day–1, respectively)] over a 10-month period. No AEs related to the study solution occurred, and lactoferrin was tolerated by each group. During lactoferrin supplementation, one bloodstream infection occurred in each group, but there were no incidences of urinary tract infections and no cases of necrotizing enterocolitis. Postnatal cytomegalovirus acquisition was detected in the group treated with 200 mg·kg–1·day–1 (n = 2). There were no adverse effects on hepatic, renal, or hematologic function. All of the patients survived to discharge. Bovine lactoferrin at doses up to 300 mg·kg–1·day–1 is safe in preterm infants. Future studies examining higher doses of lactoferrin, length of treatment, and potency of different products will aid in determining the optimal approach for the use of lactoferrin to prevent infections in preterm infants.

Background/aims This article describes the proposed design of a phase I study evaluating the safety of ceramide nanoliposome and vinblastine among an initial set of 19 possible dose combinations in patients with relapsed/refractory acute myeloid leukemia and patients with untreated acute myeloid leukemia who are not candidates for intensive induction chemotherapy. Methods Extensive collaboration between statisticians and clinical investigators revealed the need to incorporate several adaptive features into the design, including the flexibility of adding or eliminating certain dose combinations based on safety criteria applied to multiple dose pairs. During the design stage, additional dose levels of vinblastine were added, increasing the dimension of the drug combination space and thus the complexity of the problem. Increased complexity made application of existing drug combination dose-finding methods unsuitable in their current form. Results Our solution to these challenges was to adapt a method based on isotonic regression to meet the research objectives of the study. Application of this adapted method is described herein, and a simulation study of the design’s operating characteristics is conducted. Conclusion The aim of this article is to bring to light examples of novel design applications as a means of augmenting the implementation of innovative designs in the future and to demonstrate the flexibility of adaptive designs in satisfying changing design conditions.

Acute kidney injury (AKI) increases the risk for chronic kidney disease (CKD). However, there are few tools to detect microstructural changes after AKI. Here, cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) was applied to examine the heterogeneity of kidney pathology in the transition from AKI to CKD. Adult male mice received folic acid followed by cationic ferritin and were euthanized at four days (AKI), four weeks (CKD-4) or 12 weeks (CKD-12). Kidneys were examined by histologic methods and CFE-MRI. In the CKD-4 and CKD-12 groups, glomerular number was reduced and atubular cortical lesions were observed. Apparent glomerular volume was larger in the AKI, CKD-4 and CKD-12 groups compared to controls. Glomerular hypertrophy occurred with ageing. Interglomerular distance and glomerular density were combined with other MRI metrics to distinguish the AKI and CKD groups from controls. Despite significant heterogeneity, the noninvasive (MRI-based) metrics were as accurate as invasive (histological) metrics at distinguishing AKI and CKD from controls. To assess the toxicity of cationic ferritin in a CKD model, CKD-4 mice received cationic ferritin and were examined one week later. The CKD-4 groups with and without cationic ferritin were similar, except the iron content of the kidney, liver, and spleen was greater in the CKD-4 plus cationic ferritin group. Thus, our study demonstrates the accuracy and safety of CFE-MRI to detect whole kidney pathology allowing for the development of novel biomarkers of kidney disease and providing a foundation for future in vivo longitudinal studies in mouse models of AKI and CKD to track nephron fate.

BACKGROUND: Rhinovirus frequently causes asthma exacerbations among children and young adults who are allergic. The interaction between allergen and rhinovirus-induced symptoms and inflammation over time is unclear. OBJECTIVE: Our aim was to compare the response to an experimental inoculation with rhinovirus-16 in allergic asthmatics with the response in healthy controls and to evaluate the effects of administrating omalizumab before and during the infection. METHODS: Two clinical trials were run in parallel. In one of these trials, the response to an experimental inoculation with rhinovirus-16 among asthmatics with high levels of total IgE was compared to the response in healthy controls. The other trial compared the effects of administering omalizumab versus placebo to asthmatics in a randomized, double-blind placebo-controlled investigation. The primary outcome for both trials compared lower respiratory tract symptoms (LRTSs) between study groups over the first 4 days of infection. RESULTS: Frequent comparisons of symptoms, lung function, and blood eosinophil counts revealed differences that were more pronounced among allergic asthmatics than among controls by days 2 and 3 after virus inoculation. Additionally, an augmentation of upper respiratory tract symptom scores and LRTS scores occurred among the atopic asthmatics versus the controls during the resolution of symptoms (P < .01 for upper respiratory symptom tract scores and P < .001 for LRTS scores). The beneficial effects of administering omalizumab on reducing LRTSs and improving lung function were strongest over the first 4 days. CONCLUSIONS: LRTSs and blood eosinophil counts were augmented and lung function was reduced among allergic asthmatics early after rhinovirus inoculation but increased late in the infection during symptom resolution. The effect of administering omalizumab on the response to rhinovirus was most pronounced during the early/innate phase of the infection.

We hypothesize that addition of illustrated handouts during prenatal consultations decreases maternal anxiety and improves maternal knowledge. Inpatient gravid women at 25 0/7–34 6/7 weeks gestation were randomized to Standard or Illustrated consults, verbal consults supplemented with a visual handout. Post consult surveys were administered assessing maternal anxiety and knowledge acquisition. We enrolled 82 women; 54 to Standard Consult, 28 to Illustrated Consult. Consult duration was the same across arms. Anxiety and knowledge were not impacted by the intervention overall. We found higher mean knowledge by 17% for consults ≥31 min (P = 0.006; 95% CI 0.67–3.82), and 13% in primigravids (P = 0.032; 95% CI 0.15–3.21) in the intervention arm. Using illustrated handouts is feasible and does not increase duration of prenatal consults. It may improve knowledge acquisition in long consults and in primigravida women, although it does not impact anxiety and knowledge overall.

Immunotherapies targeting the PD-1/PD-L1 pathway have shown some success in cervical and vulvar squamous cell carcinomas, but little is known about the potential vulnerability of these tumors to other checkpoint inhibitors. TIM-3 is a checkpoint molecule that exerts immunosuppressive function via its interaction with Gal-9. TIM-3 and Gal-9 have been identified on a variety of malignancies but have not been studied in cervical and vulvar cancers, nor has their relationship to PD-L1 been established. Sixty-three cervical and vulvar invasive (n = 34) and intraepithelial lesions (n = 29) were assessed for TIM-3, Gal-9, and PD-L1 in tumor/lesional cells and associated immune cells. Tumoral TIM-3 expression was identified in 85% of squamous cell carcinomas but only 21% of intraepithelial lesions (p

Background Hematology-oncology patients often require blood and blood product transfusions, including platelets (PLTs), to maintain stability. Administering PLTs in a shorter timeframe may prove beneficial by possibly raising platelet counts to a higher level faster, and allowing patients to be disconnected from IV pumps sooner. Objective To evaluate the optimal (safe and effective) transfusion time by comparing standard administration of PLTs over 2–4 h to the investigational administration of PLTs over 30–45 min in the pediatric hematology-oncology inpatient population. Methodology A pilot trial was conducted using a convenience sample of hematology-oncology children. Children prescribed a PLT transfusion while admitted to an inpatient unit were eligible. If randomized to the intervention group, the nurse administered the PLTs over 30–45 min. If randomized to the standard group, the nurse administered the PLTs over 2–4 h. Post transfusion PLTcount was drawn 30 min after completion. The child was monitored closely for adverse reactions. Results Eleven participants were enrolled in the study and 20 PLT infusions administered. No adverse events were noted. There was not a significant difference in changes in PLT counts by group (post minus pre), p = 0.082. There was not a significant difference in post infusion PLT counts, p = 0.727. There was a significant difference in the rate of change in PLT counts by groups, p = 0.003. Nursing implications This pilot study provides preliminary evidence that PLTs may be safely and effectively administered over 30–45 min in pediatric hematology-oncology patients. With quicker PLT administration, patients can be disconnected from IV pumps sooner.

Objective: In this descriptive study, we evaluated perceptions and knowledge of inpatient glycemic control among resident physicians. Methods: We performed this study at four academic medical centers: the University of Mississippi Medical Center, University of Virginia Health System, University of Louisville Health Sciences Center, and Emory University. We designed a questionnaire, and Institutional Review Board approval was granted at each institution prior to study initiation. We then administered the questionnaire to Internal Medicine and Medicine-Pediatric resident physicians. Results: A total of 246 of 438 (56.2%) eligible resident physicians completed the Inpatient Glycemic Control Questionnaire (IGCQ). Most respondents (85.4%) reported feeling comfortable treating and managing inpatient hyperglycemia, and a majority (66.3%) agreed they had received adequate education. Despite self-reported comfort with knowledge, only 51.2% of respondents could identify appropriate glycemic targets in critically ill patients. Only 45.5% correctly identified appropriate inpatient random glycemic target values in noncritically ill patients, and only 34.1% of respondents knew appropriate preprandial glycemic targets in noncritically ill patients. A small majority (54.1%) were able to identify the correct fingerstick glucose value that defines hypoglycemia. System issues were the most commonly cited barrier to successful inpatient glycemic control. Conclusion: Most respondents reported feeling comfortable managing inpatient hyperglycemia but had difficulty identifying appropriate inpatient glycemic target values. Future interventions could utilize the IGCQ as a pre- and postassessment tool and focus on early resident education along with improving system environments to aid in successful inpatient glycemic control. Abbreviations: DM = diabetes mellitus; Emory = Emory University Healthcare; IGC = inpatient glycemic control; IGCQ = Inpatient Glycemic Control Questionnaire; IRB = Institutional Review Board; PGY = postgraduate year; UMMC = University of Mississippi Medical Center; UVA = University of Virginia Health System; UL = University of Louisville Health Sciences Center

IntroductionWe previously reported the physical, psychological and behavioral 3-month post-treatment results of a randomized controlled trial comparing glycemic excursion minimization (GEM) versus conventional weight loss (WL) therapy in the management of type 2 diabetes (T2D). GEM is a paradigm shift in the lifestyle management of T2D that focuses on reducing postnutrient glucose excursions, rather than reducing weight. We now present the 13-month follow-up results.Research design and methodsThe initial study sample of 172 were 30–80 years old, had T2D for ≤10 years, an HbA1c ≥6.8% (51 mmol/mol), and were not using insulin. Participants were randomized to 6 hours of group treatment, either to WL or one of three versions of GEM. GEM groups differed in degree of blood glucose (BG) feedback provided during treatment: no recommended feedback, systematic capillary BG feedback before and after nutrient intake and physical activity, or continuous glucose monitoring. Since these GEM groups did not differ in pre-post improvement they were combined for initial and current analyses. Of those who completed the 3-month postassessment, 100% and 96% of the WL and GEM participants completed the 13-month follow-up assessment.ResultsPre to follow-up within-group comparisons indicated WL participants sustained improvement in body mass index (BMI) (−0.9±1.4, p=0.001). GEM participants continued to benefit in their HbA1c (−0.5±1.4, pConclusionsWhile WL sustained improvement in BMI, GEM sustained benefits across a broad range of physical, behavioral and psychological parameters, beneficial for clinicians and adults with T2D. This may be especially relevant for primary care physicians who manage about 90% of patients with T2D.Trial registration numberNCT03196895.

Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

OBJECTIVES With the Children with Hemiparesis Arm and Hand Movement Project (CHAMP) multisite factorial randomized controlled trial, we compared 2 doses and 2 constraint types of constraint-induced movement therapy (CIMT) to usual customary treatment (UCT). METHODS CHAMP randomly assigned 118 2- to 8-year-olds with hemiparetic cerebral palsy to one of 5 treatments with assessments at baseline, end of treatment, and 6 months posttreatment. Primary blinded outcomes were the assisting hand assessment; Peabody Motor Development Scales, Second Edition, Visual Motor Integration; and Quality of Upper Extremity Skills Test Dissociated Movement. Parents rated functioning on the Pediatric Evaluation of Disabilities Inventory-Computer Adaptive Test Daily Activities and Child Motor Activity Log How Often scale. Analyses were focused on blinded and parent-report outcomes and rank-order gains across all measures. RESULTS Findings varied in statistical significance when analyzing individual blinded outcomes. parent reports, and rank-order gains. Consistently, high-dose CIMT, regardless of constraint type, produced a pattern of greatest short- and long-term gains (1.7% probability of occurring by chance alone) and significant gains on visual motor integration and dissociated movement at 6 months. O’Brien’s rank-order analyses revealed high-dose CIMT produced significantly greater improvement than a moderate dose or UCT. All CIMT groups improved significantly more in parent-reported functioning, compared with that of UCT. Children with UCT also revealed objective gains (eg, 48% exceeded the smallest-detectable assisting hand assessment change, compared with 71% high-dose CIMT at the end of treatment). CONCLUSIONS CHAMP provides novel albeit complex findings: although most individual blinded outcomes fell below statistical significance for group differences, high-dose CIMT consistently produced the largest improvements at both time points. An unexpected finding concerns shifts in UCT toward higher dosages, with improved outcomes compared with previous reports.

BACKGROUND: Hyperglycemia is common after acute ischemic stroke and is associated with worse outcome, but intensive glucose control has not improved outcome. There is also a racial disparity in outcome after stroke, with Black patients more likely to have functional impairment than whites. We aimed to evaluate if there were racial differences in outcomes in acute ischemic stroke patients treated with intensive glucose control. METHODS: We performed a post-hoc analysis of the Stroke Hyperglycemia Insulin Network Effort (SHINE) trial to determine if Black patients had worse functional outcome than whites and if standard versus intensive glucose control modified that association. We included non-Hispanic white and Black patients. The primary outcome was excellent functional outcome (90-day modified Rankin Score of 0-1). To account for patient clustering by study site, we fit mixed-effects logistic regression models to our outcome and tested the interaction of treatment and race. RESULTS: We included 895 patients, of which 304 (34%) were Black and 591 (66%) were white. The rate of excellent outcome was 31.6% in Black patients versus 41.0% in white patients (p=0.006). After adjusting for potential confounders, the odds ratio for excellent outcome in Black patients was 0.54 (95% CI 0.38-0.77). The interaction term between treatment and race was significant (p=0.067). In the intensive treatment arm, Black patients had a predicted probability of excellent outcome of 26.4% (20.1-32.8) versus 42.7% (37.6-47.9) for white patients (p

Background: Accumulating evidence supports an association between nephron number and susceptibility to kidney disease. However, it is not currently possible to directly measure nephron number in a clinical setting. Recent clinical studies have used glomerular density from a single biopsy and whole kidney cortical volume from imaging to estimate both nephron number and single nephron glomerular filtration rate. However, the accuracy of these estimates from individual subjects is unknown. Furthermore, it is not clear how sample size or biopsy location may influence these estimates. These questions are critical to study design and to the potential translation of these tools to estimate nephron number in individual subjects. Methods: We measured the variability in estimated nephron number derived from needle or virtual biopsies and cortical volume in human kidneys declined for transplantation. We performed multiple needle biopsies in the same kidney, and examined the three-dimensional spatial distribution of nephron density by magnetic resonance imaging. We determined the accuracy of a single kidney biopsy to predict the mean nephron number estimated from multiple biopsies from the same kidney. Results: A single needle biopsy had a 15% chance and virtual biopsy had a 60% chance of being within 20% of whole kidney nephron number. Single needle biopsies could be used to detect differences in nephron number between large cohorts of several hundred subjects. Conclusions: The number of subjects required to accurately detect differences in nephron number between populations can be predicted based on natural intra-kidney variability in glomerular density. A single biopsy is insufficient to accurately predict nephron number in individual subjects.

This paper studies the notion of coherence in interval-based dose-finding methods. An incoherent decision is either: (i) a recommendation to escalate the dose following an observed dose-limiting toxicity, or (ii) a recommendation to de-escalate the dose following a non-dose-limiting toxicity. In a simulated example, we illustrate that the Bayesian optimal interval method and the Keyboard method are not coherent. We generated dose-limiting toxicity outcomes under an assumed set of true probabilities for a trial of n = 36 patients in cohorts of size 1, and we counted the number of incoherent dosing decisions that were made throughout this simulated trial. Each of the methods studied resulted in 13/36 (36%) incoherent decisions in the simulated trial. Additionally, for two different target dose-limiting toxicity rates, 20% and 30%, and a sample size of n = 30 patients, we randomly generated 100 dose-toxicity curves and tabulated the number of incoherent decisions made by each method in 1000 simulated trials under each curve. For each method studied, the probability of incurring at least one incoherent decision during the conduct of a single trial is greater than 75%. Coherency is an important principle in the conduct of dose-finding trials. Interval-based methods violate this principle for cohorts of size 1 and require additional modifications to overcome this shortcoming. Researchers need take a closer look at the dose assignment behavior of interval-based methods when using them to plan dose-finding studies.

Objective This study aimed to determine if delayed cord clamping (DCC) is associated with a reduction in neonatal acute kidney injury (AKI). Study Design A retrospective single-center cohort study of 278 very low birth weight (VLBW) neonates was performed to compare the incidence of AKI in the following groups: immediate cord clamping (ICC), DCC, and umbilical cord milking. AKI was diagnosed by the modified neonatal Kidney Diseases and Improving Global Outcomes (KDIGO) definition. Results The incidence of AKI in the first week was 20.1% with no difference between groups (p = 0.78). After adjustment for potential confounders, the odds of developing AKI, following DCC, compared with ICC was 0.93 (confidence interval [CI]: 0.46–1.86) with no reduction in the stage of AKI between groups. Conclusion In this study, DCC was not associated with a reduced rate of AKI in VLBW neonates. However, the data suggest that DCC is also not harmful to the kidneys, further supporting the safety of DCC in VLBW neonates.

BACKGROUND Children with hypoplastic left heart syndrome (HLHS) have risk for mortality and/or transplantation. Previous studies have associated right ventricular (RV) indices in a single echocardiogram with survival, but none have related serial measurements to outcomes. This study sought to determine whether the trajectory of RV indices in the first year of life was associated with transplant-free survival to stage 3 palliation (S3P). METHODS HLHS patients at a single center who underwent stage 1 palliation (S1P) between 2000 and 2015 were reviewed. Echocardiographic indices of RV size and function were obtained before and following S1P and stage 2 palliation (S2P). The association between these indices and transplant-free survival to S3P was examined. RESULTS There were 61 patients enrolled in the study with 51 undergoing S2P, 20 S3P, and 18 awaiting S3P. In the stage 1 perioperative period, indexed RV end-systolic area increased in patients who died or needed transplant following S2P, and changed little in those surviving to S3P (3.37 vs -0.04 cm2 /m2 , P = .017). Increased indexed RV end-systolic area was associated with worse transplant-free survival. (OR = 0.815, P = .042). In the interstage period, indexed RV end-diastolic area increased less in those surviving to S3P (3.6 vs 9.2, P = .03). CONCLUSION Change in indexed RV end-systolic area through the stage 1 perioperative period was associated with transplant-free survival to S3P. Neither the prestage nor poststage 1 indexed RV end-systolic area was associated with transplant-free survival to S3P. Patients with death or transplant before S3P had a greater increase in indexed RV end-diastolic area during the interstage period. This suggests earlier serial changes in RV size which may provide prognostic information beyond RV indices in a single study.

Background: Most women in the United States do not meet their breastfeeding goals, and low-income women breastfeed at lower rates than the general population. While risk factors for early cessation have been documented, specific reasons for discontinuing among this population are less understood. We examined reasons for cessation among low-income mothers to inform the development of targeted strategies to address breastfeeding disparities. Materials and Methods: We performed a secondary data analysis using prospective data collected during a randomized intervention trial of Special Supplemental Nutrition Program for Women, Infants, and Children (WIC)-eligible women interviewed in the third trimester and at 1, 3, and 6 months postpartum. We included the 221 women who initiated breastfeeding and stopped by 6 months. Women's reasons for discontinuing breastfeeding were grouped by thematic category and compared by time of breastfeeding cessation. Results: The most common reasons reported overall for breastfeeding cessation were concerns about breast milk supply and latch difficulty. Some reasons differed significantly by time of cessation. Latch difficulty was reported most often by women who breastfed for 1 month or less; supply concerns increased with increasing breastfeeding duration. Returning to work/school was uncommonly reported for those who stopped by 1 month, but more frequently reported in those with later cessation. Conclusions: We found that low-income women reported similar reasons for early breastfeeding cessation as have been reported for other populations of women. These results underscore the need for appropriately timed, culturally sensitive interventions to reduce disparities in duration of breastfeeding, specifically to address latch difficulty in the first few weeks and supply concerns as infants grow.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Purpose: Many of the therapeutic agents that are being used currently were developed using the 3+3 decision rule for dose finding. Over the past 30 years, several dose-finding designs have been proposed and evaluated, including the “continual reassessment method” (CRM) and the “Bayesian optimal interval design” (BOIN). This research investigates the role of the choice of an early-phase design on the likelihood that drugs entering the drug development pipeline will have 2 successful phase III trials. Experimental Design: Using simulation, each agent in a population of hypothetical agents was tracked through the drug development process, from initial dose finding to 2 confirmatory phase III trials. Varying the designs of the phase I, II, and III trials allows for an assessment of the effect of the choice of designs on the proportion of agents with successful phase III trials. Results: The results indicate that using the CRM or BOIN, rather than the 3+3, substantially enhances the proportion of effective agents that have successful phase III trials, with the CRM having a greater effect than BOIN. A larger phase II trial magnifies the effect of the phase I design. Conclusions: The results underscore the importance of the choice of the early-phase designs. Use of the 3+3 results in fewer agents with successful phase III trials compared with the CRM or BOIN. The difference is more pronounced among highly effective agents. In addition, the results show the importance of a sufficiently powered phase II trial.

Introduction Efforts to lower glycosylated hemoglobin (A1c) in patients with type 2 diabetes (T2D) are intended to reduce the risk of diabetic complications, but A1c is not the only factor contributing to this risk. Consequently, we re-analyzed published data from a broad-spectrum lifestyle intervention that lowered A1c to assess its effectiveness in lowering the overall risk of two complications of T2D, namely, coronary heart disease (CHD) and stroke. Methods Data from 37 adults who participated in a randomized clinical trial of a lifestyle intervention intended to reduce postprandial glucose (PPG) were re-analyzed for their pre- and post-treatment risk of CHD and stroke using the T2D-specific UK Prospective Diabetes Study (UKPDS) v2.0 risk algorithm. Results Compared to participants who received routine care, those using the lifestyle intervention had a significantly greater reduction in 10-year risk for CHD, but not for stroke. Conclusion These secondary analyses suggest that broad-spectrum lifestyle interventions that focus on lowering PPG may lower the risk of future CHD, which could guide future research. Trial Registration ClinicalTrials.gov ID: NCT02432391.

Gastroesophageal reflux disease (GERD) in premature neonates may manifest as apnea, bradycardia, growth failure, aspiration, or feeding intolerance. Erythromycin ethylsuccinate (EES), is often used as a pro-kinetic in the management of GERD, despite lack of evidence or safety from randomized controlled trials. We sought to study the efficacy of enteral EES at a dose of 50 mg · kg · day in decreasing the frequency of gastroesophageal reflux events as determined by pH-multichannel intraluminal impedance (pH-MII) monitoring.In a randomized, double-blind, placebo-controlled trial, eligible premature neonates with clinical signs of GERD underwent 24-hour pH-MII monitoring. If5 reflux events were identified on pH-MII, then subjects were randomized to receive either EES or placebo. Repeat 24-hour pH-MII was performed on day 7 of study treatment and compared to initial pH-MII.Forty-three premature neonates were enrolled. Of those, 31 neonates were randomized, 15 to EES and 16 to placebo with a median (IQR) pretreatment total reflux events per 24 hours of 23 (16-40) and 29 (12-40), respectively. Day 7 total events per 24 hours decreased by 4 events in the EES group to 19 (15-33) and by 10 events in the placebo group to 19 (11-26) (P = 0.09). There were no differences in pretreatment and day 7 acidic and nonacidic reflux, proximal reflux, total or percent reflux time, median or longest bolus clearance time, or nurse-reported apnea events between groups.Enteral EES did not decrease reflux events on 24-hour pH-MII at the dose studied. Therefore, it may be ineffective in the treatment of GERD in premature neonates.

CD8+ T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8+ T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8+ T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8+ T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8+ T-cell dysfunction.

Background: Although rates of complementary food and beverage (CFB) consumption among infants under 4 to 6 months of age have been declining, they remain well above the American Academy of Pediatri...

Introduction: In acute ischemic stroke (AIS), hyperglycemia promotes enhanced blood brain barrier permeability, tissue acidosis, and oxygen free radicals, and may increase risk of post-tPA symptomatic intracerebral hemorrhage (sICH). We performed a pre-planned analysis from the SHINE trial (NCT01369069) to examine the effects of blood glucose (BG) control on post-thrombolysis ICH. Hypothesis: In AIS, (1) post-tPA BG measures are associated with sICH, and (2) intensive insulin therapy can reduce the risk of sICH. Methods: Hyperglycemic AIS patients Results: Of the 1151 SHINE participants, 725 (63%) received IV tPA (median age 65, 46% women, 29% Black, 18% Hispanic). Median NIHSS was 7, baseline BG 187 (IQR 153-247) mg/dL, and onset to tPA was 2.2 hrs (1.6-2.9). Post-tPA sICH occurred in 3.6% (3% intensive vs. 4.3% standard, OR 1.10, 95% CI 0.60-2.01, p=0.697). There was a consistent association between post-tPA BG measures and sICH. In the first 12 hours, every 10 mg/dL increase in median BG increased odds of sICH by ~8% (OR 1.08, 95% CI 1.03-1.14, p=0.004), and a greater percentage of BG measures 80-130 mg/dL decreased odds of sICH by ~11% (0.89, 95% CI 0.80-0.99, p=0.030). Conclusion: In this pre-planned analysis, intensive insulin therapy was not associated with a reduced risk of post-tPA sICH. However, post-tPA hyperglycemia was associated with a higher risk of sICH overall, particularly in the early post-treatment period. These data provide class IIa, level B-R evidence that post-tPA glucose levels between 80-130 mg/dL are associated with decreased risk of sICH. Acknowledgments: NIH-NINDS U01 NS069498.