Functional near-infrared spectroscopy-based prefrontal cortex oxygenation during working memory tasks in sickle cell disease.

Significance: Sickle cell disease (SCD), characterized by painful vaso-occlusive crises, is associated with cognitive decline. However, objective quantification of cognitive decline in SCD remains a challenge, and the associated hemodynamics are unknown. Aim: To address this, we utilized functional near-infrared spectroscopy (fNIRS) to measure prefrontal cortex (PFC) oxygenation responses to N-back working memory tasks in SCD patients and compared them with healthy controls. Approach: We quantified the PFC oxygenation rate as an index of cognitive activity in each group and compared them. In half of the participants, a Stroop test was administered before they started N-back to elevate their baseline stress level. Results: In SCD compared to healthy controls, we found that (1) under a high baseline stress level, there were significantly greater oxygenation responses during the 2-back task, further elevated with histories of stroke; (2) there was a marginally slower N-back response time, and it was even slower with a history of stroke; and (3) the task accuracy was not different. Conclusions: Additional requirements for processing time, PFC resources, and PFC oxygenation in SCD patients offer an important basis for understanding their cognitive decline and highlight the potential of fNIRS for evaluating cognitive functions.

Vasoconstriction Response to Mental Stress in Sickle Cell Disease: The Role of the Cardiac and Vascular Baroreflexes.

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 (MTS). We sought to determine whether this anomalous finding for MTS is related to inter-group differences in baseline cardiac and vascular autonomic function. Fifteen subjects with SCD and 15 healthy volunteers participated in three MTS tasks: N-back, Stroop, and pain anticipation (PA). R-R interval (RRI), arterial blood pressure and finger photoplethysmogram (PPG) were continuously monitored before and during these MTS tasks. The magnitude of vasoconstriction was quantified using change in PPG amplitude (PPGa) 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 RRIs. The vascular baroreflex sensitivity (BRSv) was quantified using the same approaches, modified for application to beat-to-beat diastolic blood pressure and 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 MTS 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 PA. After adjusting for differences in BRSv within and between groups, we found no difference in the vasoconstriction responses to all three 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 vaso-occlusive crisis (VOC) to be triggered by exposure to stressful events.

Mental stress causes vasoconstriction in subjects with sickle cell disease and in normal controls.

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.

Erythrocyte and plasma oxidative stress appears to be compensated in patients with sickle cell disease during a period of relative health, despite the presence of known oxidative agents.

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 (200 µg/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.

Sickle Cell Disease Subjects Have a Distinct Abnormal Autonomic Phenotype Characterized by Peripheral Vasoconstriction With Blunted Cardiac Response to Head-Up Tilt.

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.

Early Use of Anti-influenza Medications in Hospitalized Children With Tracheostomy.

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.

A novel cross-correlation methodology for assessing biophysical responses associated with pain.

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.

Upper Airway Narrowing during Central Apnea in Obese Adolescents.

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; r2 = 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.

A Retrospective Review of Infants Receiving Sildenafil.

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 (<1.5 mg/kg/day), medium (1.5-3.75 mg/kg/day), large (3.76-7.5 mg/kg/day), and very large (>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.

Individuals with sickle cell disease have a significantly greater vasoconstriction response to thermal pain than controls and have significant vasoconstriction in response to anticipation of pain.

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 < .01). Although subjective measures, pain threshold and pain tolerance were not different between SCD subjects and controls, but the vaso-reactivity index characterizing the microvascular blood flow response to painful stimuli was significantly higher in SCD patients (P = .0028). This global vasoconstriction increases microvascular transit time, and may promote entrapment of sickle cells in the microvasculature, making vaso-occlusion more likely. The rapidity of the global vasoconstriction response indicates a neural origin that may play a part in the transition from steady-state to VOC, and may also contribute to the variability in VOC frequency observed in SCD patients.

Biophysical markers of the peripheral vasoconstriction response to pain in sickle cell disease.

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.

Empirical model of human blood transverse relaxation at 3 T improves MRI T2 oximetry.

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.

A physician survey reveals differences in management of idiopathic pulmonary hemosiderosis.

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.

Autonomic responses to cold face stimulation in sickle cell disease: a time-varying model analysis.

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.

Chronic transfusion therapy improves but does not normalize systemic and pulmonary vasculopathy in sickle cell disease.

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.

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker.

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.

Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer.

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 IgG(3) 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.

A Ras activation pathway dependent on Syk phosphorylation of protein kinase C.

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.