Roberta M. Kato

Tec Kinases Mediate Sustained Calcium Influx via Site-specific Tyrosine Phosphorylation of the Phospholipase Cγ Src Homology 2-Src Homology 3 Linker

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) 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 PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Brutons 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 PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. 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, Tyr753 and Tyr759. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγ2 carboxyl-terminal sites, Tyr1197 and Tyr1217, 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 PLCγ2 SH2-SH3 linker.

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, FcεRI. 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 PKCβI and PKCα, respectively, are phosphorylated by Syk in the membrane compartment of FcεRI-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 FcεRI-stimulated mast cells.

PKC-|[beta]| controls I|[kappa]|B kinase lipid raft recruitment and activation in response to BCR signaling

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

Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum.

Significance A major challenge in treating chronic infections is the lack of insight into microbial survival mechanisms in vivo. Many drugs require cells to be doubling rapidly to have their greatest effect, yet the in vivo pathogen growth rate is largely unknown. By labeling freshly expectorated mucus from cystic fibrosis patients with heavy water, we found that the effective growth rates of Staphylococcus aureus are at least two orders of magnitude slower, on average, than typically studied in the laboratory, and are extremely heterogeneous at the single-cell level. These findings underscore the need to study slow growth physiology to gain insight into pathogen survival mechanisms, motivated by the hope that such insight will ultimately help improve drug design and clinical outcomes. 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.

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.

Peripheral Vasoconstriction and Abnormal Parasympathetic Response to Sighs and Transient Hypoxia in Sickle Cell Disease

RATIONALE Sickle cell disease is an inherited blood disorder characterized 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 between transient 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 in microvascular perfusion; however, it induced substantial transient 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. Although the sigh frequency was the same in both groups, the probability of a sigh inducing a perfusion drop was 78% in patients with sickle cell and 17% in control subjects (P < 0.001). Evidence for sigh-induced sympathetic nervous 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 enhanced autonomic nervous system–mediated sigh–vasoconstrictor response in patients with sickle cell that could increase red cell retention in the microvasculature, promoting vasoocclusion.

Pulmonary function abnormalities in childhood cancer survivors treated with bleomycin.

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.

Patients with sickle cell anemia on simple chronic transfusion protocol show sex differences for hemodynamic and hematologic responses to transfusion

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.

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

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).

Sputum Glucose and Glycemic Control in Cystic Fibrosis-Related Diabetes: A Cross-Sectional Study

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 (HbA1c) > 6.5% have worse pulmonary function, longer and more frequent exacerbations and also higher sputum glucose levels than patients with HbA1c ≤ 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 HbA1c 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 HbA1c > 6.5% were hospitalized on average 6 days longer than those with HbA1c ≤ 6.5% (p < 0.01). Current clinical care guidelines for cystic fibrosis-related diabetes target HbA1c ≤ 7% to limit long-term microvascular damage, but more stringent glycemic control (HbA1c ≤ 6.5%) may further reduce the short-term pulmonary complications.