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Low forced vital capacity (FVC) is associated with decreased exercise capacity in CHD. Multiple prior cardiac surgeries have been associated with low FVC. We seek to understand the relationship between low FVC, number of cardiac surgeries and cardiopulmonary response leading to decreased exercise capacity. Retrospective chart review of demographics, surgical history and exercise testing including spirometry was performed in patients with CHD. Single ventricle patients were excluded. Low FVC was defined as a Z-score below the lower limit of normal. Exercise parameters were expressed as a percent of predicted. There were 93 patients with 2 ventricle CHD identified over 34 months with cardiopulmonary exercise testing. The FVC Z-score directly correlated with peak VO2% (R2 = 0.07, p

Vaso-occlusive crisis (VOC) is a hallmark of sickle cell disease (SCD) and occurs when deoxygenated sickled red blood cells occlude the microvasculature. Any stimulus, such as mental stress, which decreases microvascular blood flow will increase the likelihood of red cell entrapment resulting in local vaso-occlusion and progression to VOC. Neurally mediated vasoconstriction might be the physiological link between crisis triggers and vaso-occlusion. In this study, we determined the effect of mental stress on microvascular blood flow and autonomic nervous system reactivity. Sickle cell patients and controls performed mentally stressful tasks, including a memory task, conflict test and pain anticipation test. Blood flow was measured using photoplethysmography, autonomic reactivity was derived from electrocardiography and perceived stress was measured by the State-Trait Anxiety Inventory questionnaire. Stress tasks induced a significant decrease in microvascular blood flow, parasympathetic withdrawal and sympathetic activation in all subjects. Of the various tests, pain anticipation caused the highest degree of vasoconstriction. The magnitude of vasoconstriction, sympathetic activation and perceived stress was greater during the Stroop conflict test than during the N-back memory test, indicating the relationship between magnitude of experimental stress and degree of regional vasoconstriction. Baseline anxiety had a significant effect on the vasoconstrictive response in sickle cell subjects but not in controls. In conclusion, mental stress caused vasoconstriction and autonomic nervous system reactivity in all subjects. Although the pattern of responses was not significantly different between the two groups, the consequences of vasoconstriction can be quite significant in SCD because of the resultant entrapment of sickle cells in the microvasculature. This suggests that mental stress can precipitate a VOC in SCD by causing neural-mediated vasoconstriction.

Recent studies have shown that individuals with sickle cell disease (SCD) exhibit greater vasoconstriction responses to physical autonomic stressors, such as heat pain and cold pain than normal individuals, but this is not the case for mental stress. We sought to determine whether this anomalous finding for mental stress is related to inter-group differences in baseline cardiac and vascular autonomic function. Fifteen subjects with SCD and 15 healthy volunteers participated in 3 mental stress tasks: N-back, Stroop, and pain anticipation. R-R interval, arterial blood pressure and finger photoplethysmogram (PPG) were continuously monitored before and during these mental stress tasks. The magnitude of vasoconstriction was quantified using change in PPG amplitude from the baseline period. To represent basal autonomic function, we assessed both cardiac and vascular arms of the baroreflex during the baseline period. Cardiac baroreflex sensitivity (BRSc) was estimated by applying both the “sequence” and “spectral” techniques to beat-to-beat measurements of systolic blood pressure and R-R intervals. The vascular baroreflex sensitivity (BRSv) was quantified using the same approaches, modified for application to beat-to-beat diastolic blood pressure and PPG amplitude (PPGa) measurements. Baseline BRSc was not different between SCD and non-SCD subjects, was not correlated with BRSv, and was not associated with the vasoconstriction responses to mental stress tasks. BRSv in both groups was correlated with mean PPGa, and since both baseline PPGa and BRSv were lower in SCD, these results suggested that the SCD subjects were in a basal state of higher sympathetically-mediated vascular tone. In both groups, baseline BRSv was positively correlated with the vasoconstriction responses to N-back, Stroop and pain anticipation. After adjusting for differences in BRSv within and between groups, we found no difference in the vasoconstriction responses to all 3 mental tasks between SCD and non-SCD subjects. The implications of these findings are significant in subjects with SCD since vasoconstriction reduces microvascular flow and prolongs capillary transit time, increasing the likelihood for VOC to be triggered by exposure to stressful events.

Sickle cell disease (SCD) is a monogenetic disease that results in the formation of hemoglobin S. Due to more rapid oxidation of hemoglobin S due to intracellular heme and adventitious iron in SCD, it has been thought that an inherent property of SCD red cells would be an imbalance in antioxidant defenses and oxidant production. Less deformable and fragile RBC in SCD results in intravascular hemolysis and release of free hemoglobin (PFHb) in the plasma, which might be expected to produce oxidative stress in the plasma. Thus, we aimed to characterize intracellular and vascular oxidative stress in whole blood and plasma samples from adult SCD patients and controls recruited into a large study of SCD at Children's Hospital of Los Angeles. We evaluated the cellular content of metHb and several components of the antioxidant system in RBC as well as oxidation of GSH and Prx-2 oxidation in RBC after challenge with hydroperoxides. Plasma markers included PFHb, low molecular weight protein bound heme (freed heme), hemopexin, isoprostanes, and protein carbonyls. While GSH was slightly lower in SCD RBC, protein carbonyls, NADH, NAD+ and total NADP++NADPH were not different. Furthermore, GSH or Prx-2 oxidation was not different after oxidative challenge in SCD vs. Control. Elevated freed heme and PFHb had a significant negative, non-linear association with hemopexin. There appeared to be a threshold effect for hemopexin (200μg/ml), under which the freed heme rose acutely. Plasma F2-isoprostanes were not significantly elevated in SCD. Despite significant release of Hb and elevation of freed heme in SCD when hemopexin was apparently saturated, there was no clear indication of uncompensated vascular oxidative stress. This somewhat surprising result, suggests that oxidative stress is well compensated in RBCs and plasma during a period of relative health.

Alpha thalassemia is a hemoglobinopathy due to decreased production of the α-globin protein from loss of up to four α-globin genes, with one or two missing in the trait phenotype. Individuals with sickle cell disease who co-inherit the loss of one or two α-globin genes have been known to have reduced risk of morbid outcomes, but the underlying mechanism is unknown. While α-globin gene deletions affect sickle red cell deformability, the α-globin genes and protein are also present in the endothelial wall of human arterioles and participate in nitric oxide scavenging during vasoconstriction. Decreased production of α-globin due to α-thalassemia trait may thereby limit nitric oxide scavenging and promote vasodilation. To evaluate this potential mechanism, we performed flow-mediated dilation and microvascular post-occlusive reactive hyperemia in 27 human subjects (15 missing one or two α-globin genes and 12 healthy controls). Flow-mediated dilation was significantly higher in subjects with α-trait after controlling for age (P = .0357), but microvascular perfusion was not different between groups. As none of the subjects had anemia or hemolysis, the improvement in vascular function could be attributed to the difference in α-globin gene status. This may explain the beneficial effect of α-globin gene loss in sickle cell disease and suggests that α-globin gene status may play a role in other vascular diseases.

Background Two modes of ventilation commonly used in children requiring chronic home mechanical ventilation (HMV) via tracheostomy are Assist Control (AC) and Synchronized Intermittent Mandatory Ventilation with Pressure Support (SIMV+PS). There has been no study comparing these two modes of ventilation in children requiring chronic HMV. Methods We studied children requiring HMV capable of completing speech testing. Study participants were blinded to changes and studied on both modes, evaluating their oxygen saturation, end tidal carbon dioxide (PET CO2 ), heart rate, respiratory rate, and respiratory pattern. Subjects completed speech testing and answered subjective questions about their level of comfort, ease of breathing, and ease of speech. Results Fifteen children aged 12.3±4.8 years were tested. There was no difference in mean oxygen saturation, minimum oxygen saturation, mean PET CO2 , maximum PET CO2 , mean heart rate, and mean respiratory rate. The maximum heart rate on AC was significantly lower than SIMV+PS, p=0.047. Subjects breathed significantly above the set rate on SIMV+PS (p=0.029), though not on AC. Subjects found it significantly easier to speak on AC, though there was no statistically significant difference in speech testing. Four subjects had multiple prolonged PS breaths on SIMV+PS. Many subjects exhibited an abnormal cadence to speech, with some speaking during both inhalation and exhalation phases of breathing. Conclusions There were few differences between AC and SIMV+PS, with a few parameters favoring AC that may not be clinically significant. This includes subjective perception of ease of speech. We also found unnatural patterns of speech in children requiring HMV. This article is protected by copyright. All rights reserved.

Sickle cell disease (SCD) is a monogenic hemoglobinopathy associated with significant morbidity and mortality. Cardiopulmonary, vascular and sudden death are the reasons for the majority of young adult mortality in SCD. To better understand the clinical importance of multi-level vascular dysfunction, in 2009 we assessed cardiac function including tricuspid regurgitant jet velocity (TRV), tissue velocity in systole(S') and diastole (E'), inflammatory, rheologic and hemolytic biomarkers as predictors of mortality in patients with SCD. With up to 9 years of follow up, we determined survival in 95 children, adolescents and adults with SCD. Thirty-eight patients (40%) were less than 21 years old at initial evaluation. Survival and Cox proportional-hazards analysis were performed. There was 19% mortality in our cohort, with median age at death of 35 years. In the pediatric subset, there was 11% mortality during the follow up period. The causes of death included cardiovascular and pulmonary complications in addition to other end-organ failure. On Cox proportional-hazards analysis, our model predicts that a 0.1 m/s increase in TRV increases risk of mortality 3%, 1 cm/s increase in S' results in a 91% increase, and 1 cm/s decrease in E' results in a 43% increase in mortality. While excluding cardiac parameters, higher plasma free hemoglobin was significantly associated with risk of mortality (p=.049). In conclusion, elevated TRV and altered markers of cardiac systolic and diastolic function predict mortality in a cohort of adolescents and young adult patients with SCD. These predictors should be considered when counseling cardiovascular risk and therapeutic optimization at transition to adult providers.

Persons with sickle cell disease (SCD) exhibit subjective hypersensitivity to cold and heat perception in experimental settings, and triggers such as cold exposure are known to precipitate vaso-occlusive crises by still unclear mechanisms. Decreased microvascular blood flow (MBF) increases the likelihood of vaso-occlusion by increasing entrapment of sickled red blood cells in the microvasculature. Because those with SCD have dysautonomia, we anticipated that thermal exposure would induce autonomic hypersensitivity of their microvasculature with an increased propensity toward vasoconstriction. We exposed 17 patients with SCD and 16 control participants to a sequence of predetermined threshold temperatures for cold and heat detection and cold and heat pain via a thermode placed on the right hand. MBF was measured on the contralateral hand by photoplethysmography, and cardiac autonomic balance was assessed by determining heart rate variability. Thermal stimuli at both detection and pain thresholds caused a significant decrease in MBF in the contralateral hand within seconds of stimulus application, with patients with SCD showing significantly stronger vasoconstriction (P = .019). Furthermore, patients with SCD showed a greater progressive decrease in blood flow than did the controls, with poor recovery between episodes of thermal stimulation (P = .042). They had faster vasoconstriction than the controls (P = .033), especially with cold detection stimulus. Individuals with higher anxiety also experienced more rapid vasoconstriction (P = .007). Augmented vasoconstriction responses and progressive decreases in perfusion with repeated thermal stimulation in SCD are indicative of autonomic hypersensitivity in the microvasculature. These effects are likely to increase red cell entrapment in response to clinical triggers such as cold or stress, which have been associated with vaso-occlusive crises in SCD.

John Sunwoo,1 Patjanaporn Chalacheva,1 Maha Khaleel,2 Payal Shah,2 Richard Sposto,3 Roberta M Kato,4 Jon Detterich,5 Lonnie K Zeltzer,6 John C Wood,1,5 Thomas D Coates,2 Michael CK Khoo1 1Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, USA; 2Hematology Section, Children’s Center for Cancer, Blood Disease and Bone Marrow Transplantation, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 3Children’s Center for Cancer, Blood Disease and Bone Marrow Transplantation, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 4Division of Pediatric Pulmonology, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 5Division of Pediatric Cardiology, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; 6Pediatric Pain and Palliative Care Program, Department of Pediatrics, Division of Hematology-Oncology, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA, USA Purpose: The purpose of this work was to noninvasively detect and quantify microvascular blood flow changes in response to externally applied pain in humans. The responsiveness of the microvasculature to pain stimulation might serve as an objective biomarker in diseases associated with altered pain perception and dysregulated vascular functions. The availability of such a biomarker may be useful as a tool for predicting outcome and response to treatments, particularly in diseases like sickle cell anemia where clinical manifestations are directly linked to microvascular perfusion. We, therefore, developed a method to distinguish the blood flow response due to the test stimulus from the blood flow measurement that also includes concurrent flow changes from unknown origins.Subjects and methods: We measured the microvascular blood flow response in 24 healthy subjects in response to a train of randomly spaced and scaled heat pulses on the anterior forearm. The fingertip microvascular perfusion was measured using laser Doppler flowmetry. The cross-correlation between the heat pulses and the blood flow response was computed and tested for significance against the null distribution obtained from the baseline recording using bootstrapping method.Results: We estimated correlation coefficients, response time, response significance, and the magnitude of vasoreactivity from microvascular blood flow responses. Based on these pain response indices, we identified strong responders and subjects who did not show significant responses.Conclusion: The cross-correlation of a random pattern of painful stimuli with directly measured microvascular flow can detect vasoconstriction responses in a noisy blood flow signal, determine the time between stimulus and response, and quantify the magnitude of this response. This approach provided an objective measurement of vascular response to pain that may be an inherent characteristic of individual human subjects, and may also be related to the severity of vascular disorders. Keywords: objective quantification of pain, randomized heat pulse train, laser Doppler flowmetry, cross-correlation, bootstrap test, vasoreactivity

There have been no published reports of central respiratory control abnormalities in pediatric patients with UNC80 or KCNJ11 mutations which cause neurologic channelopathies. We describe an 8-year-...

OBJECTIVE The purpose of the study was to assess mortality in an infant population receiving sildenafil. METHODS A retrospective review of hospitalized infants at Children's Hospital Los Angeles who received sildenafil between 2008 and 2012 was conducted. Patient characteristics, comorbidities, and treatment characteristics were analyzed. Primary outcome was mortality at discharge. Sildenafil dosage ranges were based on the Sildenafil in Treatment-Naïve Children, Aged 1–17 Years, With Pulmonary Arterial Hypertension trial and were categorized as small (7.5 mg/kg/day). RESULTS A total of 147 infants were studied. A total of 82% of patients had severe pulmonary hypertension. Our data revealed 29% mortality at discharge. Mortality increased with increasing sildenafil dosage: 14% (small), 19% (medium), 49% (large), and 90% (very large). On multivariate analysis of sildenafil dosage, other pulmonary hypertension therapies, presence of persistent cardiac shunts, and duration of sildenafil, odds of dying were significantly higher with combined high and very high sildenafil dosage groups compared with combined low and medium dosage groups (OR, 13.2; CI, 4.4–39.5; p < 0.0001). CONCLUSION Sildenafil was given to critically ill infants with multiple risk factors for mortality. Although higher doses cannot be causally related to mortality, there appears to be no added benefit by escalating the sildenafil dose.

Introduction Absence of alpha globin genes has long been known to influence the physiology of sickle cell disease (SCD). Individuals with SCD who are missing one or two alpha globin genes have decreased rates of cerebral vasculopathy, stroke, acute chest syndrome, and leg ulcers (Bernaudin, Blood 2008; Flanagan, Blood 2011; Nolan, Br J Haematol 2006). Although there is laboratory evidence of decreased hemolytic rate in these patients (Higgs, N Engl J Med 1982), the mechanism for their improved clinical outcomes has not been identified. Recently, the alpha globin protein has been shown to be present in the endothelial wall of human arterioles, where it modulates nitric oxide (NO) scavenging during vasoconstriction (Straub, Nature 2012). In mice, pharmacological inhibition of alpha globin leads to increased endothelial NO activity, independently of NO production, and results in increased blood perfusion, reduced systemic hypertension, and increased pulmonary artery vasodilation (Keller, Hypertension 2016; Alvarez, Am J Respir Cell Mol Biol 2017). The relationship between absence of alpha globin and arterial vasodilation, and the role of alpha globin in NO-mediated vascular signaling are potential mechanisms that could explain the beneficial effect of missing alpha globin genes in SCD. Using alpha thalassemia as a naturally occurring human model of alpha globin gene knockout, we hypothesized that loss of alpha globin genes leads to improvement in microvascular blood flow in thalassemia trait subjects without hemolysis. Methods Alpha thalassemia trait subjects missing one or two alpha globin genes, and healthy controls were recruited to the study, which was approved by the Children's Hospital Los Angeles Institutional Review Board. Blood samples were obtained from all subjects to test for hemoglobin, mean corpuscular volume (MCV), reticulocyte count, plasma hemoglobin, lactate dehydrogenase, and alpha globin genotype. We assessed flow-mediated dilation (FMD) of the brachial artery following distal forearm occlusion (Detterich, Blood 2015) simultaneously with laser Doppler flowmetry (LDF) and photoplethysmography (PPG) in the fingertip. We also measured the increase in microvascular perfusion with a thermal stimulus. The maximal change in vascular perfusion after provocation indicates vasodilatory capacity. Statistical analysis was performed in JMP® version 14 (SAS Institute Inc., USA). Results Twenty-seven subjects were enrolled, including 12 controls (4 alpha globin genes), 10 patients with 3 alpha globin genes and 5 with 2. The mean MCV was lower in subjects missing alpha globin genes than in controls (p=0.0099). Importantly, hemoglobin levels and markers of hemolysis were normal in both groups. There was no detectable difference in FMD between individuals missing one and two alpha globin genes; thus, these groups were combined and labeled as alpha trait for further analyses. FMD was significantly higher in alpha trait subjects after adjusting for age (Figure 1, p=0.0357). Missing alpha globin genes had no effect on microvascular flow by LDF or PPG (data not shown). Discussion FMD is an established and specific predictor of NO bioavailability (Thijssen, Am J Physiol Heart Circ Physiol 2011), and, in addition to shear-mediated NO circulation in conduit vessels, it reflects the sum of flow in multiple arteriolar networks downstream of the conduit artery. Using this method, a difference in endothelial function between control and alpha thalassemia trait was easily detected (Figure 1). Because endothelial alpha globin is present in arterioles rather than conduit vessels (Butcher, Free Radic Biol Med 2014), we measured microvascular flow in a 1-mm3 volume in the skin using a laser Doppler sensor, and in the fingertip by PPG, but were unable to detect an effect of alpha trait. As none of the subjects had anemia or evidence of hemolysis, the significantly increased FMD associated with loss of alpha globin genes is most likely due to increased NO as a result of decreased scavenging by alpha globin. The finding reported here that lower alpha globin gene number is associated with increased NO-related perfusion in humans may explain the beneficial effect of alpha thalassemia trait in SCD and suggests that the presence of alpha thalassemia trait may also play a role in other types of vascular disease. Disclosures Wood: BiomedInformatics: Consultancy; Imago Biosciences: Consultancy; BluebirdBio: Consultancy; Celgene: Consultancy; WorldcareClinical: Consultancy; Philips Medical Systems: Research Funding. Coates:apo pharma (Chiesi Pharma): Consultancy, Honoraria; Sangamo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios pharma: Consultancy, Honoraria; Vifor Pharma: Consultancy, Honoraria; Celgene, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

In sickle cell disease (SCD), prolonged capillary transit times, resulting from reduced peripheral blood flow, increase the likelihood of rigid red cells entrapment in the microvasculature, predisposing to vaso-occlusive crisis. Since changes in peripheral flow are mediated by the autonomic nervous system (ANS), we tested the hypothesis that the cardiac and peripheral vascular responses to head-up tilt (HUT) are abnormal in SCD. Heart rate, respiration, non-invasive continuous blood pressure and finger photoplethysmogram (PPG) were monitored before, during, and after HUT in SCD, anemic controls and healthy subjects. Percent increase in heart rate from baseline was used to quantify cardiac ANS response, while percent decrease in PPG amplitude represented degree of peripheral vasoconstriction. After employing cluster analysis to determine threshold levels, the HUT responses were classified into four phenotypes: (CP) increased heart rate and peripheral vasoconstriction; (C) increased heart rate only; (P) peripheral vasoconstriction only; and (ST) subthreshold cardiac and peripheral vascular responses. Multinomial logistic regression (MLR) was used to relate these phenotypic responses to various parameters representing blood properties and baseline cardiovascular activity. The most common phenotypic response, CP, was found in 82% of non-SCD subjects, including those with chronic anemia. In contrast, 70% of SCD subjects responded abnormally to HUT: C-phenotype = 22%, P-phenotype = 37%, or ST-phenotype = 11%. MLR revealed that the HUT phenotypes were significantly associated with baseline cardiac parasympathetic activity, baseline peripheral vascular variability, hemoglobin level and SCD diagnosis. Low parasympathetic activity at baseline dramatically increased the probability of belonging to the P-phenotype in SCD subjects, even after adjusting for hemoglobin level, suggesting a characteristic autonomic dysfunction that is independent of anemia. Further analysis using a mathematical model of heart rate variability revealed that the low parasympathetic activity in P-phenotype SCD subjects was due to impaired respiratory-cardiac coupling rather than reduced cardiac baroreflex sensitivity. By having strong peripheral vasoconstriction without compensatory cardiac responses, P-phenotype subjects may be at increased risk for vaso-occlusive crisis. The classification of autonomic phenotypes based on HUT response may have potential use for guiding therapeutic interventions to alleviate the risk of adverse outcomes in SCD.

BACKGROUND: Early administration of anti-influenza medications is recommended for all children hospitalized with influenza. We investigated whether early use of anti-influenza medications is associated with improved outcomes in children with tracheostomy hospitalized with influenza. METHODS: We performed a multicenter retrospective cohort study through the Pediatric Health Information System database for patients aged 30 days to 19 years who were discharged between October 1, 2007, and September 30, 2015 with diagnostic codes for both influenza and tracheostomy. Our primary predictor was receipt of anti-influenza medications on hospital day 0 or 1. We used propensity score matching to adjust for confounding by indication. Primary outcomes were length of stay (LOS) and 30-day all-cause revisit rate (emergency department visit or hospital admission). RESULTS: Of 1436 discharges screened, 899 met inclusion criteria. The median admission age was 5 years (interquartile range: 2–10). The majority had multiple complex chronic conditions (median 3; interquartile range: 3–4) and technology dependence, such as gastrostomy tube (73.6%). After matching 772 unique admissions by propensity score, LOS was shorter for the cohort receiving early anti-influenza medications (6.4 vs 7.5 days; P = .01) without increase in revisit rate (27.5% vs 24.1%; P = .28). More than 80% in both cohorts received empirical antibiotics, and the duration of antibiotic therapy was similar (5.0 vs 5.6 days; P = .11). CONCLUSIONS: Early use of anti-influenza medications in children with tracheostomy hospitalized with influenza is associated with shorter LOS, but these children continue to receive antibiotics despite identification and treatment of their viral infections.

We have previously demonstrated that sickle cell disease (SCD) patients maintain normal global systemic and cerebral oxygen delivery by increasing cardiac output. However, ischemic end-organ injury remains common suggesting that tissue oxygen delivery may be impaired by microvascular dysregulation or damage. To test this hypothesis, we performed fingertip laser Doppler flowmetry measurements at the base of the nailbed and regional oxygen saturation (rSO2 ) on the dorsal surface of the same hand. This was done during flow mediated dilation (FMD) studies in 26 chronically transfused SCD, 75 non-transfused SCD, and 18 control subjects. Chronically transfused SCD patients were studied prior to and following a single transfusion and there was no acute change in rSO2 or perfusion. Laser Doppler estimates of resting perfusion were 76% higher in non-transfused and 110% higher in transfused SCD patients, compared to control subjects. In contrast, rSO2 was 12 saturation points lower in non-transfused SCD patients, but normal in the transfused SCD patients. During cuff occlusion, rSO2 declined at the same rate in all subjects suggesting similar intrinsic oxygen consumption rates. Upon cuff release, laser doppler post occlusive hyperemia was blunted in SCD patients in proportion to their resting perfusion values. Transfusion therapy did not improve the hyperemia response. FMD was impaired in SCD subjects but partially ameliorated in transfused SCD subjects. Taken together, non-transfused SCD subjects demonstrate impaired conduit artery FMD, impaired microcirculatory post-occlusive hyperemia, and resting hypoxia in the hand despite compensated oxygen delivery, suggesting impaired oxygen supply-demand matching. Transfusion improves FMD and oxygen supply-demand matching but not microcirculation hyperemic response.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography–mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.IMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Purpose We sought a human blood T2-oximetery calibration curve over the wide range of hematocrits commonly found in anemic patients applicable with T2 relaxation under spin tagging (TRUST). Methods Blood was drawn from five healthy control subjects. Ninety-three in vitro blood transverse relaxation (T2b) measurements were performed at 37°C over a broad range of hematocrits (10–55%) and oxygen saturations (14–100%) at 3 Tesla (T). In vivo TRUST was performed on 35 healthy African American control subjects and 11 patients with chronic anemia syndromes. Results 1/T2 rose linearly with hematocrit (r2 = 0.96), for fully saturated blood. Upon desaturation, 1/T2 rose linearly with the square of the oxygen extraction, (1-Y)2, and the slope was linearly proportional to hematocrit (r2 = 0.88). The resulting bilinear model between 1/T2, (1-Y)2, and hematocrit had a combined r2 of 0.96 and a coefficient of variation of 6.1%. Using the in vivo data, the bilinear model had significantly lower bias and variability than existing calibrations, particularly for low hematocrits. In vivo Bland Altman analysis demonstrated clinically relevant bias that was −6% (absolute saturation) for hematocrits near 30% and rose to + 6% for hematocrits near 45%. Conclusion This work introduces a robust bilinear calibration model that should be used for MRI oximetry. Magn Reson Med 77:2364–2371, 2017. © 2016 International Society for Magnetic Resonance in Medicine

Rationale: The use of real-time magnetic resonance imaging (MRI) for the evaluation during sleep-related respiratory events can lead to better understanding of airway dynamics. Objectives: To investigate the dynamic anatomy of the upper airway during central apnea. Methods: The study included obese adolescents who snore and were otherwise healthy. Subjects underwent an overnight baseline polysomnogram. Subjects slept during a 24-minute real-time upper airway MRI scan wearing a full face mask attached to a pneumotach. Sleep versus wakefulness during the MRI was inferred from the heart rate and respiratory patterns. Central apneas were scored using tracings of facemask airflow and abdominal bellows. The cross-sectional area of the upper airway before, during, and after each central apnea event was recorded. Results: Eight subjects were studied and 57 central apnea events were observed during real-time MRI scanning during natural sleep. The median age of subjects was 14.0 years (interquartile range [IQR], 13.5 to 15.5). The median average reduction in cross-sectional area during central apnea events was −38% (IQR, −27 to −51) for primary snorers and −45% (IQR, −40 to −54) for subjects with obstructive sleep apnea. The percentage decrease in cross-sectional area of upper airway during a central apnea event was positively correlated to the length of the central apnea (ρ = 0.389; r(2) = 0.152; P = 0.003). Conclusions: We observed that there is upper airway narrowing during central apneas during natural sleep in obese adolescent subjects, using real-time MRI.

Tricuspid regurgitant (TR) jet velocity and its relationship to pulmonary hypertension has been controversial in sickle cell disease (SCD). Plasma free hemoglobin is elevated in SCD patients and acutely impairs systemic vascular reactivity. We postulated that plasma free hemoglobin would be negatively associated with both systemic and pulmonary endothelial function, assessed by flow-mediated dilation (FMD) of the brachial artery and TR jet velocity, respectively. Whole blood viscosity, plasma free hemoglobin, TR jet, and FMD were measured in chronically transfused SCD pre- and posttransfusion (N = 25), in nontransfused SCD (N = 26), and in ethnicity-matched control subjects (N = 10). We found increased TR jet velocity and decreased FMD in nontransfused SCD patients compared with the other 2 groups. TR jet velocity was inversely correlated with FMD. There was a striking nonlinear relationship between plasma free hemoglobin and both TR jet velocity and FMD. A single transfusion in the chronically transfused cohort improved FMD. In our patient sample, TR jet velocity and FMD were most strongly associated with plasma free hemoglobin and transfusion status (transfusions being protective), and thus consistent with the hypothesis that intravascular hemolysis and increased endogenous erythropoiesis damage vascular endothelia.

The painful vaso-occlusive crises (VOC) that characterize sickle cell disease (SCD) progress over hours from the asymptomatic steady-state. SCD patients report that VOC can be triggered by stress, cold exposure and pain itself. We anticipated that pain could cause neural-mediated vasoconstriction, decreasing regional blood flow and promoting entrapment of sickle cells in the microvasculature. Therefore, we measured microvascular blood flow in the fingers of both hands using plethysmography and laser-Doppler flowmetry while applying a series of painful thermal stimuli on the right forearm in 23 SCD patients and 25 controls. Heat pain applied to one arm caused bilateral decrease in microvascular perfusion. The vasoconstriction response started before administration of the thermal pain stimulus in all subjects, suggesting that pain anticipation also causes significant vasoconstriction. The time delay between thermal pain application and global vasoconstriction ranged from 5 to 15.5 seconds and increased with age (p

Background Bleomycin is associated with pulmonary toxic side effects including pneumonitis and pulmonary fibrosis. We evaluated the prevalence of long-term pulmonary function abnormalities in children receiving bleomycin therapy in the context of current chemotherapeutic regimens. Methods A retrospective review of patients who received bleomycin between January 1999 and December 2011 was conducted. Abnormalities in the most recent pulmonary function test (PFT) at least 1 year after diagnosis were analyzed. Results Two-hundred and seven patients had received bleomycin. The results of PFT performed at least 1 year from diagnosis were available for 80 patients. Median time of follow up was 3.9 years (range 1.1–11.76 years). Median cumulative dose of bleomycin was 65 IU/m2 (range 10–120). The most common diagnoses were Hodgkin lymphoma and germ cell tumor. At least one pulmonary function abnormality was present in 42 (52.5%) patients. When classified in groups, 22.5% patients had obstructive lung disease, 7.5% had restrictive lung disease, 28.8% had hyperinflation and 14% of patients had non-uniform distribution of ventilation. Non-Hispanic patients (OR 2.81) and children younger than 8 years (OR 4.14) had higher odds of having an abnormal PFT parameter. Very few patients had pulmonary symptoms. Conclusions More than half the patients who received bleomycin had subclinical pulmonary dysfunction as evidenced by abnormalities in pulmonary function tests, although the incidence of clinical symptoms was low. Pediatr Blood Cancer 2014;61:1679–1684. © 2014 Wiley Periodicals, Inc.

Studies have demonstrated an association between abnormal spirometry (AS) and decreased exercise capacity among single ventricle (1V) circulation. This association is unclear in two ventricle (2V) circulation. We aim to study patients undergoing cardiopulmonary exercise testing (CPET) to evaluate

Background Chronic transfusion therapy (CTT) is a mainstay for stroke prophylaxis in sickle cell anemia, but its effects on hemodynamics are poorly characterized. Transfusion improves oxygen-carrying capacity, reducing demands for high cardiac output, while decreasing hemoglobin (Hb)S%, reticulocyte count, and hemolysis. We hypothesized that transfusion would improve oxygen-carrying capacity, but that would be counteracted by a decrease in cardiac output due to increased hematocrit (Hct) and vascular resistance, leaving oxygen delivery unchanged. Study Design and Methods To test this hypothesis, we examined patients on CTT immediately before transfusion and again 12 to 120 hours after transfusion, using echocardiography and near infrared spectroscopy. Results Comparable increases in Hb and Hct and decreases in reticulocyte count and HbS with transfusion were observed in all patients, but males had a larger rebound of HbS%, reticulocyte count, and free Hb levels between transfusions. In males, transfusion decreased heart rate by 12%, stroke volume by 15%, and cardiac index by 24% while estimates for pulmonary and systemic vascular resistance increased, culminating in 6% decrease in oxygen delivery. In contrast, stroke volume and cardiac index and systemic and pulmonary vascular resistance did not change in women after transfusion, such that oxygen delivery improved 17%. Conclusion In our sample population, males exhibit a paradoxical reduction in oxygen delivery in response to transfusion because the increase in vascular resistance is larger than the increase in oxygen capacity. This may result from an inability to adequately suppress their HbS% between transfusion cycles.

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

Rationale:Sicklecelldiseaseisaninheritedblooddisordercharacterized by vasoocclusive crises. Although hypoxia and pulmonary disease are known risk factors for these crises, the mechanisms that initiate vasoocclusive events are not well known. Objectives: To study the relationship betweentransient hypoxia, respiration, and microvascular blood flow in patients with sickle cell. Methods: We established a protocol that mimics nighttime hypoxic episodes and measured microvascular blood flow to determine if transient hypoxia causes a decrease in microvascular blood flow. Significant desaturations were induced safely by five breaths of 100% nitrogen. Measurements and Main Results: Desaturation did not induce change inmicrovascularperfusion;however,itinducedsubstantialtransient parasympathetic activity withdrawal in patients with sickle cell disease, but not controls subjects. Marked periodic drops in peripheral microvascular perfusion, unrelated to hypoxia, were triggered by sighs in 11 of 11 patients with sickle cell and 8 of 11 control subjects. Althoughthesigh frequencywasthesameinbothgroups,theprobability of a sigh inducing a perfusion drop was 78% in patients with sickle cell and 17% in control subjects(P ,0.001). Evidence for sighinduced sympatheticnervous system dominance was seen in patients with sickle cell (P , 0.05), but was not significant in control subjects. Conclusions: These data demonstrate significant disruption of autonomic nervous system balance, with marked parasympathetic withdrawal in response to transient hypoxia. They draw attention to an enhancedautonomicnervous system–mediatedsigh–vasoconstrictor response in patients with sickle cell that could increase red cell retention in the microvasculature, promoting vasoocclusion.

This study compared pulmonary function tests (PFTs) with cardiac, pancreatic and liver iron in 76 thalassemia major (TM) patients. Restrictive lung disease was observed in 16%, hyperinflation in 32%, and abnormal diffusing capacity in 3%. While no patients met Global Initiative for Chronic Lung Disease criteria for airways obstruction, there were indicators of small airways disease and air trapping. PFTs did not correlate with somatic iron burden, blood counts or haemolysis. Restrictive lung disease was associated with inflammation. We conclude that TM patients have pulmonary abnormalities consistent with small airways obstruction. Restrictive disease and impaired diffusion are less common.

Introduction: Vaso-occlusive pain crisis (VOC) is a significant contributor to the morbidity of Sickle Cell Disease (SCD) and cold exposure has long been associated with increased frequency and intensity of VOC. However, the mechanism by which cold exposure causes the transition from steady state to vaso-occlusion has not been well elucidated. Decreased regional blood flow results in red blood cells spending a longer period of time in the deoxygenated state in the capillaries, increasing the likelihood of hemoglobin S polymerization in the microvasculature and subsequent vaso-occlusion. Regional blood flow is primarily regulated by the autonomic nervous system (ANS) and recent literature shows evidence of cardiovascular autonomic dysfunction in SCD and of its role as a disease modulating factor. We hypothesized that cold exposure triggers a central autonomic response leading to vasoconstriction in the microvasculature and that SCD subjects will have a stronger response when compared to controls. Methods: 17 SCD and 16 control (healthy or sickle trait) subjects aged 13 to 39 years were exposed to thermal stimuli via a computer-controlled thermode (TSA-II) placed on the thenar eminence of the right hand. Predetermined individual threshold temperatures for heat detection, cold detection, heat pain and cold pain were applied, and changes in the microvascular blood flow (MBF) were measured at a sub-second sampling rate on the contralateral thumb using photo-plethysmography (PPG). Mean MBF was derived from the amplitude of the PPG signal, a greater decrease in mean MBF from baseline signifies stronger vasoconstriction. The vasoconstriction response within the PPG signal, and the time (in sec) to vasoconstriction were determined from the cross-correlation function of the pain stimulus signal and the vascular response. The R-to-R interval (RRI) derived from electrocardiogram was used to evaluate cardiac autonomic balance. Standard deviation of RRI represents total heart rate variability, Spectral indices of the RRI represent parasympathetic activity (high frequency power; HFP) and sympatho-vagal balance (Low to high Ratio; LHR = low frequency power(LFP)/HFP). Prior to the study, subjects were administered measures of current anxiety and general level of anxiety (STAI Y1 and Y2, respectively) as well as a measure of pain related anxiety (PASS). Results: All the thermal stimulation tasks caused a significant decrease in MBF from baseline (p values for cold detection, heat detection, heat pain and cold pain=0.027, At baseline and across all tasks, SCD subjects showed a significantly lower heart rate variability suggestive of autonomic dysfunction (standard deviation of RRI- 41.2 Vs 50 msec, p=0.007). Both cold and heat pain caused a significant increase in the mean LFP and LHR from baseline with no significant differences in HFP thus suggesting an overall increase in the cardiac sympathetic output with thermal pain. Conclusion: Using MBF as an objective marker of pain responses, cold pain causes significant decrease in MBF followed by other thermal stimuli. The rapid vasoconstriction responses to the thermal stimuli occurred in the contralateral hand, suggesting a neural mechanism. SCD subjects demonstrated an augmented peripheral vasoconstriction response with evidence of cardiac autonomic imbalance. Our findings imply that global autonomic mediated vasoconstriction and subsequent vaso-occlusion is the likely mechanistic link between cold exposure and VOC in SCD. Thus, vascular autonomic reactivity is a biomarker of basic SCD pathophysiology and serves as a potential target for prevention and treatment of pain in SCD. Disclosures Coates: ApoPharma: Consultancy, Honoraria; Sangamo: Consultancy, Honoraria; Vifor Pharma: Consultancy; Celgene Corp.: Consultancy.

Introduction Over the last two decades, sickle cell disease has become a model of diffuse endotheliopathy. In 2004, Gladwin and colleagues demonstrated that elevated tricuspid regurgitation jet velocity (TRJ) was an independent predictor of mortality and that chronic red blood cell hemolysis underlies pulmonary vascular disease (Gladwin et. al. NEJM, PMID:14985486). Belhassen and colleagues demonstrated that flow-mediated dilation of the brachial artery (FMD) was decreased in patients with sickle cell disease, specifically shear-mediated vasodilation (Belhassen et. al. Blood, PMID:11238095). Our group demonstrated that plasma free hemoglobin links decreased FMD and elevated TRJ in patients with sickle cell disease and that chronic transfusion therapy improves FMD (Detterich et. al. Blood, PMID:26036801). We aimed to determine whether markers of vascular function predict the risk of mortality in a cohort of patients with sickle cell disease. Methods This was a single center, prospective cohort study of non-transfused and chronically transfused sickle cell anemia patients at Children's Hospital of Los Angeles between 2009 and 2012 aiming to assess vascular function in sickle cell disease. This study was approved by the Children's Hospital Los Angeles Institutional Review Board. We used FMD, a test of shear-mediated endothelial nitric oxide release as the model for vascular function. We simultaneously measured the regional hemoglobin oxygen saturation in the hand using near infrared spectroscopy (NIRS). We also measured microcirculatory perfusion using laser doppler flowmetry (LDF) over the skin of the nailbed. Echocardiography was performed on the same day to assess tricuspid regurgitation jet velocity and cardiac function. Markers of inflammation, cell count and hemolysis were also measured on the day of the study. We prospectively followed them until 2018 to assess our primary outcome, survival. All of the measurements were made pre-transfusion for chronically transfused subjects. Kaplan Meier and Cox-Proportional Hazards multivariate model was used to determine independent predictors of survival. All statistics were performed using Stata/IC version 14.0. Results We enrolled 26 chronically transfused patients with SCD and 63 non-transfused SCD patients in a study assessing the effects of hemolysis on endothelial function. Overall mortality in our cohort was 20% with a median follow up time of 7.8 years (7.1-8.3 IQR). Median age was 24 years at the time of study (17-36 IQR). Median age at the time of death was 36 years(28-51 IQR) and at the last follow up for the alive patients was 30 years (25-41 IQR), p=0.01. In the patients who died, they had higher TRJ (median 270 vs 235, p=0.03) and had elevated plasma Hgb, median 26 vs 14, p=0.01. FMD, regional oxygen saturation and microcirculatory perfusion were not different between alive and deceased patients. There was no difference for inflammatory markers (white blood cell count, platelet count, high sensitivity C-reactive protein), hemoglobin S%/transfusion, or anemia (hemoglobin/hematocrit/reticulocytes) between alive or deceased patients. By Kaplan Meier analysis individual factors associated with mortality were TRJ>250 (p=0.04) and age>21 (p=0.02) (Fig 1 and Fig 2). By Cox-Proportional Hazards Model, both TR jet velocity and plasma hemoglobin remain significant after correcting for age (Table 1). Conclusions In our cohort, there are multiple abnormal markers of vascular function and elevated markers of hemolysis; however, the strongest predictors of mortality were age, plasma free hemoglobin and tricuspid regurgitation jet velocity. These findings support the association between hemolysis, pulmonary vascular disease and mortality, a controversial topic in sickle cell disease. Disclosures Coates: Vifor Pharma: Consultancy; Sangamo: Consultancy, Honoraria; Celgene Corp.: Consultancy; ApoPharma: Consultancy, Honoraria.

Sickle cell disease (SCD) is characterized by sudden onset of painful vaso-occlusive crises (VOC), which occur on top of the underlying chronic blood disorder. The mechanisms that trigger VOC remain elusive, but recent work suggests that autonomic dysfunction may be an important predisposing factor. Heart-rate variability has been employed in previous studies, but the derived indices have provided only limited univariate information about autonomic cardiovascular control in SCD. To circumvent this limitation, a time-varying modeling approach was applied to investigate the functional mechanisms relating blood pressure (BP) and respiration to heart rate and peripheral vascular resistance in healthy controls, untreated SCD subjects and SCD subjects undergoing chronic transfusion therapy. Measurements of respiration, heart rate, continuous noninvasive BP and peripheral vascular resistance were made before, during and after the application of cold face stimulation (CFS), which perturbs both the parasympathetic and sympathetic nervous systems. Cardiac baroreflex sensitivity estimated from the model was found to be impaired in nontransfused SCD subjects, but partially restored in SCD subjects undergoing transfusion therapy. Respiratory-cardiac coupling gain was decreased in SCD and remained unchanged by chronic transfusion. These results are consistent with autonomic dysfunction in the form of impaired parasympathetic control and sympathetic overactivity. As well, CFS led to a significant reduction in vascular resistance baroreflex sensitivity in the nontransfused SCD subjects but not in the other groups. This blunting of the baroreflex control of peripheral vascular resistance during elevated sympathetic drive could be a potential factor contributing to the triggering of VOC in SCD.

Background Idiopathic pulmonary hemosiderosis (IPH) is a rare disorder of unknown etiology characterized by chronic pulmonary hemorrhage and presents with a triad of anemia, hemoptysis and pulmonary infiltrates. IPH is a diagnosis of exclusion with a variable and disparate clinical course. Despite existing therapies, few children achieve full remission while others have recurrent hemorrhage, progressive lung damage, and premature death. Methods We surveyed physicians who care for patients with IPH via a web-based survey to assess the most common practices. 88 providers responded, caring for 274 IPH patients from five continents. Results 63.3 % of respondents had patients that were initially misdiagnosed with anemia (60.0 %) or gastrointestinal bleed (18.2 %). Respondents varied in diagnostic tools used for evaluation. The key difference was in the use of lung biopsy (51.9 %) for diagnosis. Common medications respondents used for treatment at initial presentation and chronic maintenance therapy were corticosteroids (98.7 and 84.0 %, initial and chronic therapy respectively), hydroxychloroquine (33.3 and 64.0 %), azathioprine (8.0 and 37.3 %), and cyclophosphamide (4.0 and 16.0 %). There was agreement on the use of corticosteroids for exacerbation amongst all respondents. Reported deaths before adulthood occurred in 7.3 % of patients. We conclude that there were common features and specific variations in physician management of IPH. Respondents were divided on whether to perform lung biopsy for diagnosis. Conclusion Despite the availability of various immunomodulators, corticosteroids remained the primary therapy. We speculate that the standardization of care for diffuse alveolar hemorrhage will improve patient outcomes.

Cystic fibrosis-related diabetes affects up to half of cystic fibrosis patients and is associated with increased mortality and more frequent pulmonary exacerbations. However, it is unclear to what degree good glycemic control might mitigate these risks and clinical outcomes have not previously been studied in relation to glucose from the lower airways, the site of infection and CF disease progression. We initially hypothesized that diabetic cystic fibrosis patients with glycosylated hemoglobin (HbA_(1c)) > 6.5% have worse pulmonary function, longer and more frequent exacerbations and also higher sputum glucose levels than patients with HbA_(1c) ≤ 6.5% or cystic fibrosis patients without diabetes. To test this, we analyzed spontaneously expectorated sputum samples from 88 cystic fibrosis patients. The median sputum glucose concentration was 0.70 mM (mean, 4.75 mM; range, 0-64.6 mM). Sputum glucose was not correlated with age, sex, body mass index, diabetes diagnosis, glycemic control, exacerbation frequency or length, or pulmonary function. Surprisingly, sputum glucose was highest in subjects with normal glucose tolerance, suggesting the dynamics of glycemic control, sputum glucose and pulmonary infections are more complex than previously thought. Two-year mean HbA_(1c) was positively correlated with the length of exacerbation admission (p < 0.01), and negatively correlated with measures of pulmonary function (p < 0.01). While total number of hospitalizations for exacerbations were not significantly different, subjects with an HbA_(1c) > 6.5% were hospitalized on average 6 days longer than those with HbA_(1c) ≤ 6.5% (p < 0.01). Current clinical care guidelines for cystic fibrosis-related diabetes target HbA_(1c) ≤ 7% to limit long-term microvascular damage, but more stringent glycemic control (HbA_(1c) ≤ 6.5%) may further reduce the short-term pulmonary complications.

X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, near absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk- and Tec-deficient mice (BtkTec–/–) as a model for XLA, we determined if Btk gene therapy could correct this disorder. Bone marrow (BM) from 5-fluorouracil (5FU)–treated BtkTec–/– mice was transduced with a retroviral vector expressing human Btk and transplanted into BtkTec–/– recipients. Mice engrafted with transduced hematopoietic cells exhibited rescue of both primary and peripheral B-lineage development, recovery of peritoneal B1 B cells, and correction of serum immunoglobulin M (IgM) and IgG3 levels. Gene transfer also restored T-independent type II immune responses, and B-cell antigen receptor (BCR) proliferative responses. B-cell progenitors derived from Btk-transduced stem cells exhibited higher levels of Btk expression than non-B cells; and marking studies demonstrated a selective advantage for Btk-transduced B-lineage cells. BM derived from primary recipients also rescued Btk-dependent function in secondary hosts that had received a transplant. Together, these data demonstrate that gene transfer into hematopoietic stem cells can reconstitute Btk-dependent B-cell development and function in vivo, and strongly support the feasibility of pursuing Btk gene transfer for XLA.

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcepsilonRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk in the membrane compartment of FcepsilonRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcepsilonRI-stimulated mast cells.

NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.

Painful vaso-occlusive crisis (VOC), a complication of sickle cell disease (SCD), occurs when sickled red blood cells obstruct flow in the microvasculature. We postulated that exaggerated sympathetically mediated vasoconstriction, endothelial dysfunction and the synergistic interaction between these two factors act together to reduce microvascular flow, promoting regional vaso-occlusions, setting the stage for VOC. We previously found that SCD subjects had stronger vasoconstriction response to pulses of heat-induced pain compared to controls but the relative degrees to which autonomic dysregulation, peripheral vascular dysfunction and their interaction are present in SCD remain unknown. In the present study, we employed a mathematical model to decompose the total vasoconstriction response to pain into: 1) the neurogenic component, 2) the vascular response to blood pressure, 3) respiratory coupling and 4) neurogenic-vascular interaction. The model allowed us to quantify the contribution of each component to the total vasoconstriction response. The most salient features of the components were extracted to represent biophysical markers of autonomic and vascular impairment in SCD and controls. These markers provide a means of phenotyping severity of disease in sickle-cell anemia that is based more on underlying physiology than on genotype. The marker of the vascular component (BMv) showed stronger contribution to vasoconstriction in SCD than controls (p = 0.0409), suggesting a dominant myogenic response in the SCD subjects as a consequence of endothelial dysfunction. The marker of neurogenic-vascular interaction (BMn-v) revealed that the interaction reinforced vasoconstriction in SCD but produced vasodilatory response in controls (p = 0.0167). This marked difference in BMn-v suggests that it is the most sensitive marker for quantifying combined alterations in autonomic and vascular function in SCD in response to heat-induced pain.

Case: A 3-year-old boy presented to an outside hospital’s emergency department with 3 days of worsening nonproductive cough. At the outside hospital, he was found to be hypoxic with a chest radiograph demonstrating bilateral infiltrates. He was transferred to our children’s hospital with a presumed diagnosis of pneumonia. On admission to our hospital, the patient was afebrile, pale, and in mild respiratory distress. Review of systems revealed low-grade fevers for 3 days before admission but no weight loss, vomiting, hemoptysis, or sick contacts. Medical history revealed a previous diagnosis of iron-deficiency anemia, which had resolved with ferrous sulfate administration. Family history was positive for asthma. Initial oxygen saturation was 88%, which recovered to 95% on 2 L/min of oxygen by nasal cannula. His heart rate was 138 beats per minute, and his respiratory rate was 44 breaths per minute; his blood pressure was 101/66 mm Hg. The patient’s weight was 13.2 kg (10th–25th percentile), and his height was 91.5 cm (5th–10th percentile). On examination, pallor and subcostal and supraclavicular retractions were appreciated; auscultation of his lungs revealed slightly reduced breath sounds throughout, without wheeze or crackles. The rest of the physical examination was normal. The patient was continued on oxygen by nasal cannula and was started on intravenous fluids for poor oral intake secondary to respiratory effort. He was also given ceftriaxone as treatment for presumed community-acquired pneumonia (CAP). Question: What further evaluation is indicated in the case of presumed pneumonia in a patient presenting with hypoxia and respiratory distress? Discussion: According to 2011 Infectious Diseases Society of America guidelines for CAP, posteroanterior and lateral chest radiographs should be obtained in all patients who are admitted for CAP to document infiltrates and to screen for complications of pneumonia, including parapneumonic effusion, necrotizing pneumonia, and pneumothorax.1 The results of radiographs …

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

Introduction: Sickle cell disease (SCD), an inherited blood disorder, is characterized by episodes of painful vaso-occlusive crises (VOC). Peripheral vasoconstriction may contribute to VOC by prolonging the transit time of red blood cells through the microvasculature. We recently demonstrated that heat-induced pain produces stronger vasoconstriction in SCD subjects versus controls, suggesting abnormal autonomic regulation of regional blood flow in SCD. Thus, although pain is generally thought to be a consequence of VOC, it is possible that pain itself may trigger a cascade of events that leads to large-scale VOC by promoting regional peripheral vasoconstriction. Objective: To determine if the underlying functional mechanisms of the vasoconstriction response to heat-induced pain in SCD differ from normal controls. Experimental Protocols: 22 SCD and 23 control (healthy and sickle cell trait) subjects were recruited at Children's Hospital of Los Angeles. Quasi-periodic pulses of pain were delivered on the right forearm using TSA-II neuro analyzer heating thermode. Electrocardiogram, respiration, continuous blood pressure and photoplethysmogram (PPG) were recorded. Both blood pressure and PPG were measured on the ring finger and thumb on the contralateral hand. Reductions in PPG amplitude were taken to represent vasoconstriction. Analysis: A mathematical model was applied to the data to extract the key parameters relating the thermal (pain) pulses, blood pressure and respiration to vasoconstriction. The model enabled the vasoconstriction response to pain to be decomposed into: 1) a neurogenic component, measuring the direct effect of the thermal pain pulse on vasoconstriction; 2) a local vascular component, relating fluctuations in blood pressure, induced by pain, to vasoconstriction; 3) a neurogenic-vascular interaction component, reflecting the modulation of the vascular component by the pain stimulus; and 4) a respiratory coupling component. Results: The model predicted stronger vasoconstriction responses to heat pain in SCD than controls (p = 0.002), consistent with the previous results reported by Khaleel et al. (Blood 126 (23):67, 2015). The pattern of the neurogenic response in both groups was similar but stronger in SCD (Fig 1a). The time-course of the local vascular component was quite pronounced in SCD (Fig 1b) - increases in blood pressure promoted stronger and more rapid vasoconstriction in SCD relative to controls. The neurogenic-vascular interaction (not shown) caused further peripheral vasoconstriction in SCD, but tended to reduce vasoconstriction in controls. The respiratory contribution was small in both groups. Discussion & Conclusions: Heat-induced pain produces peripheral vasoconstriction via direct autonomic nervous system activation, as well as indirectly through the local vascular response to pain-triggered blood pressure fluctuations. The complex dynamics of the vasoconstriction response can only be understood by application of a mathematical model incorporating several relevant simultaneously measured and frequently sampled physiological signals. The stronger neurogenic response in SCD suggests elevated sympathetic activity compared to controls. The local vascular component in the SCD subjects exhibits a higher reactivity to increases in local blood pressure that promotes vasoconstriction, consistent with underlying endothelial dysfunction. The contribution of neurogenic-vascular interaction derived from the model likely represents the degree to which sympathetic overactivity leads to vascular dysfunction in SCD. These results suggest that dysautonomia and its interaction with peripheral vascular dysregulation participate in the genesis of vaso-occlusive crisis. Figure 1 (a) average neurogenic response (% change from baseline) to a heat pain pulse (20 °C, 10-sec long) in SCD (N = 22) and controls (N = 23); (b) average vascular response (% change from baseline) to a blood pressure pulse induced by pain (10 mmHg, 10-sec long) in SCD and controls. Figure 1. (a) average neurogenic response (% change from baseline) to a heat pain pulse (20 °C, 10-sec long) in SCD (N = 22) and controls (N = 23); (b) average vascular response (% change from baseline) to a blood pressure pulse induced by pain (10 mmHg, 10-sec long) in SCD and controls. Disclosures Wood: Biomed Informatics: Consultancy; AMAG: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Biomed Informatics: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy.

BACKGROUND: Sickle cell disease is an inheritable hemoglobinopathy that causes increased red blood cell (RBC) stiffness due to hemoglobin polymerization at low oxygen tension. This leads to microcirculatory occlusion as well as chronic hemolysis and chronic inflammation, resulting in a diffuse vasculopathy of bothmacrovascular and microvascular beds. Chronic transfusion therapy is used for both primary and secondary prevention of ischemic stroke, amacrovascular complication; however, there is limited knowledge of its effect on microvascular function. OBJECTIVE: To determine the effects of chronic transfusion therapy on microvascular function and tissue oxygenation. DESIGN/METHODS: We utilized a forearm, arterial and venous occlusion model, commonly used for flow-mediated dilation of the brachial artery, to simultaneously assess post occlusive reactive hyperemia (PORH) in the microcirculation and post hyperemic change in tissue oxygenation. PORH was measured using laser Doppler flowmetry placed over the third finger, proximal to the nailbed. Regional tissue oxygenation (rSO2) was measured using near infrared spectroscopy (NIRS) placed on the dorsal surface of the hand. FMD was measured using a linear ultrasound probe. Transfused patients were measured before and after a single transfusion. Kruskal-Wallis was used to test between group differences. Multivariate linear regression was used to model predictors of vascular function. RESULTS: We enrolled 18 control (CTL) patients, 75 non-transfused (NTR) SCD patients, and 26 chronically transfused (TR) SCD patients. Consistent with our previously published data, FMD was improved but not normalized by chronic transfusion and it was predicted by ln[plasma hgb], age and sex. PORH was reduced in both SCD patient groups when compared to the control group while resting flow by laser Doppler was elevated in both SCD groups (figure 1). PORH was also inversely related to resting flow, suggesting that basal conditions in the microcirculation are significant predictors of their response to forearm occlusion. Percent change in rSO2 was not different between the three groups. Lower baseline rSO2 was inversely associated with percent rSO2 increase following hyperemia; and, the baseline rSO2 was significantly decreased in the non-transfused SCD patients but normalized in the transfused SCD patients (Figure 2). Resting hypoxia was best predicted by RBC deformability in the non-transfused patients, even after correction for hemoglobin level, whereas resting hemoglobin levels predicted hypoxia in the transfused and healthy patients. DISCUSSION: With the goal to better understand the mechanism by which transfusion therapy decreases the risk of stroke, we tested the response to arterial and venous occlusion, simultaneously, at three different functional levels of the vascular system: 1.Conduit artery, 2.Pre-capillary arteriolar and capillary, 3.Tissue oxygen exchange. We confirm our previous findings that conduit vessel dysfunction is highly dependent on free hemoglobin, which is consistent with the hemolysis paradigm as a mechanism for sickle cell vasculopathy. This study extends our model to the microcirculation. Resting microcirculatory flow is increased in both transfused and non-transfused SCD patients; and while the resting rSO2 is near normal in the transfused SCD patients it is significantly lower in the non-transfused subjects. This creates the potential for severe hypoxia in the setting of a limited ability to augment flow through the microcirculation during periods of high demand, as in PORH. If there is also decreased conduit vessel reactivity upstream this creates the scenario over which large areas of tissue may rapidly become ischemic. This is a novel system, with simultaneous measurements in multiple vascular beds, allowing us to evaluate supply-demand matching. CONCLUSIONS: Chronic transfusion therapy improves both brachial artery endothelial function and regional tissue oxygenation but it does not significantly improve microcirculatory function, which may be near its maximal flow at rest. Hemoglobin determines resting tissue saturation in healthy and transfused SCD patients, but high shear RBC deformability determines rSO2 in non-transfused SCD patients. Figure 1. Laser Doppler resting flow is higher but post occlusive hyperemia is lower in SCD Figure 1. Laser Doppler resting flow is higher but post occlusive hyperemia is lower in SCD Figure 2. NIRS repsonse to forearm occlusion. Figure 2. NIRS repsonse to forearm occlusion. Disclosures Wood: World Care Clinical: Consultancy; Vifor: Consultancy; AMAG: Consultancy; Ionis Pharmaceuticals: Consultancy; Biomed Informatics: Consultancy; World Care Clinical: Consultancy; Ionis Pharmaceuticals: Consultancy; Apopharma: Consultancy; Vifor: Consultancy; Apopharma: Consultancy; AMAG: Consultancy; Biomed Informatics: Consultancy; Celgene: Consultancy; Celgene: Consultancy.

Introduction: Sickle cell disease (SCD) is a genetic disorder characterized by sudden onset of painful vaso-occlusive episodes (VOC) which can be triggered by stress as reported by sickle cell patients. The exact mechanism of VOC origin is not well understood; however, it could result as a progression of microvascular blockade with rigid sickled red blood cells (RBC). Anything that decreases regional blood flow (RBF) will increase the transit time that it takes the RBC to escape the microvasculature before it gets rigid and entrapped, thus increasing the chances of vaso-occlusion that may progress to VOC. The RBF is controlled by the autonomic nervous system (ANS) which is modulated by stress, and as we have previously shown, SCD patients have augmented autonomic mediated vasoconstriction response (VCR) to sigh and pain. Our studies showed significant VCR when subjects were told they were about to experience pain, suggesting that VCR might be the physiologic link between common VOC triggers like stress and sickled RBC retention in the microvasculature. Objectives: To study the effect of mental stress on autonomic parameters peripheral blood flow (PBF) and heart rate variability (HRV) in SCD. Methods: 19 SCD and 16 control (healthy and sickle cell trait) subjects were studied. Two standard mental stress tasks with graded levels of difficulty (N-back test and Stroop test) were presented to subjects using E-prime (Psychological software). Subjects were also exposed to a novel pain anticipation task, previously shown to induce VCR. PBF was measured using photo-plethysmography (PPG) on the left thumb. Reduction in PPG amplitude indicates vasoconstriction. Cardiac beat-to-beat variability (R-to-R interval;RRI) was extracted from electrocardiogram. ANS balance was derived from the following spectral indices of the RRI: high frequency power (HFP) ≈ parasympathetic activity, low frequency power (LFP), and the low/high ratio (LFP/HFP) representing sympathovagal balance. Average changes in PBF and HRV from the baseline were taken as the responses to the mental tasks. Results: There was a significant decrease in mean PBF, RRI and HFP during all the mental stressors (N-back test, Stroop test and pain fear) compared to the baseline (p Conclusions: Graded mental stress and fear of pain causes significant decrease in regional blood flow and parasympathetic withdrawal in SCD subjects and normal controls. The vasoconstriction response to "fear of pain" was significantly greater than the VRC caused by N-back and to a lesser extent in Stroop. The pattern of responses were not significantly different between SCD and controls however the consequences of decreased blood flow could be quite different because of the resulted entrapment of sickle cells in the microvasculature. This could explain how stress may play a role in the initiation of VOC in SCD by enhancing ANS mediated vasoconstriction, prolonging microvascular transit time and increasing the likelihood of vaso-occlusion. The 1st signal is mental tasks event marker, 2nd signal is photo-plethysmography (PPG) and the 3rd signal is heart rate variability derived from the electrocardiogram signal. Peripheral blood flow and heart rate variability during baseline and various mental tasks Peripheral blood flow and heart rate variability during baseline and various mental tasks Box plot of mean peripheral blood flow (PBF) during baseline, mental tasks and fear of pain. Box plot of mean peripheral blood flow (PBF) during baseline, mental tasks and fear of pain. Disclosures Wood: Celgene: Consultancy; AMAG: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Biomed Informatics: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; Biomed Informatics: Consultancy.

Pre-B cell receptor (pre-BCR) expression is critical for B lineage development. The signaling events initiated by the pre-BCR, however, remain poorly defined. We demonstrate that lipid rafts are the major functional compartment for human pre-B cell activation. A fraction of pre-BCR was constitutively raft associated, and receptor engagement enhanced this association. These events promoted Lyn activation and Igβ phosphorylation and led to the generation of a raft-associated signaling module composed of tyrosine phosphorylated Lyn, Syk, BLNK, PI3K, Btk, VAV, and PLCγ2. Formation of this module was essential for pre-BCR calcium signaling. Together, these observations directly link the previously identified genetic requirement for the components of this module in B lineage development with their functional role(s) in human pre-BCR signaling.

Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely define the T-cell inhibitory effects of HCQ, the authors evaluated T-cell antigen receptor (TCR) signaling events in a T-cell line pretreated with HCQ. In a concentration-dependent manner, HCQ inhibited anti-TCR–induced up-regulation of CD69 expression, a distal TCR signaling event. Proximal TCR signals, including inductive protein tyrosine phosphorylation, tyrosine phosphorylation of phospholipase C γ1, and total inositol phosphate production, were unaffected by HCQ. Strikingly, anti-TCR-crosslinking–induced calcium mobilization was significantly inhibited by HCQ, particularly at the highest concentrations tested (100 μmol/L) in both T-cell lines and primary T cells. HCQ, in a dose-dependent fashion, also reduced a B-cell antigen receptor calcium signal, indicating this effect may be a general property of HCQ. Inhibition of the calcium signal correlated directly with a reduction in the size of thapsigargin-sensitive intracellular calcium stores in HCQ-treated cells. Together, these findings suggest that disruption of TCR-crosslinking–dependent calcium signaling provides an additional mechanism to explain the immunomodulatory properties of HCQ.