The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation.

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.

Patients and caregivers play a central role in health care safety in the hospital, ambulatory care setting, and community. Despite this, interventions to promote patient engagement in safety are still underexplored. We conducted an overview of review articles on patient engagement interventions in safety to examine the current state of the evidence. Of the 2,795 references we evaluated, 52 articles met our full-text inclusion criteria for synthesis in 2018. We identified robust evidence supporting patients' self-management of anticoagulation medications and mixed-quality evidence supporting patient engagement in medication and chronic disease self-management, adverse event reporting, and medical record accuracy. Promising modes of patient engagement in safety, such as anticoagulation management and patient portal access, are not widely implemented. We discuss major implementation priorities and propose directions for future research and policy to enhance patient partnership within safety efforts.

Cytotoxic T lymphocytes (CTL) play a significant role in the clearance of respiratory syncytial virus (RSV) infection in humans and mice. Identification of class I MHC-restricted CTL epitopes is critical in elucidating mechanisms of CTL responses against viral infections. However, only four H-2d-restricted epitopes have been reported in mice. Because of the diversity of transgenic and knockout mice available to study immune responses, new epitopes in additional strains of mice must be identified. We therefore attempted to discover novel CTL epitopes in C57Bl/6 mice. Our efforts revealed a new H-2D(b)-restricted CTL epitope from the RSV M protein, corresponding to aa 187-195 (NAITNAKII). Also, M187-195-specific CTLs were activated with kinetics similar to the immunodominant BALB/c epitope, M2 82-90. This is the first RSV-specific CTL epitope described in a strain of mice other than BALB/c. Furthermore, identification of this H-2b-restricted CTL epitope provides access to genetically modified H-2b mice for more detailed studies of CTL mechanisms in RSV infection.

The intracellular serine/threonine kinase protein kinase C (PKC) has an important role in the genesis of pulmonary edema. This review discusses the PKC-mediated mechanisms that participate in the pulmonary endothelial response to agents involved in lung injury characteristic of the respiratory distress syndrome. Thus the paradigms of PKC-induced lung injury are discussed within the context of pulmonary transvascular fluid exchange. We focus on the signal transduction pathways that are modulated by PKC and their effect on lung endothelial permeability. Specifically, α-thrombin, tumor necrosis factor (TNF)-α, and reactive oxygen species are discussed because of their well-established roles in both human and experimental lung injury. We conclude that PKC, most likely PKC-α, is a primary supporter for lung endothelial injury in response to α-thrombin, TNF-α, and reactive oxygen species.

Intracellular Ca2؉ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K ؉ channels play an important role in Ca 2؉ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca 2؉ influx. Here we show that the Ca 2؉ -activated K ؉ channel, KCa3.1, which is critical for Ca 2؉ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca 2؉ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca 2؉ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.

Partial bladder outlet obstruction (PBOO) results in cellular damage due to ischemia and reperfusion injury. Our study seeks to establish how early this damage can occur and the role that nitric oxide may play in its pathophysiology. Surgical PBOO (1, 3, and 7 days) were performed on male New Zealand White rabbits. Half of the animals were premedicated for 3 days with N(G)-nitro-l-arginine methyl ester(l-NAME), an inhibitor of nitric oxide synthase before obstruction. Bladder weight increased with duration of PBOO but was significantly lower at 3 and 7 days in animals treated with l-NAME compared with their untreated counterparts. Contractile function decreased progressively with PBOO duration. At 1 day postobstruction, bladder contractility was significantly lower in the l-NAME rabbits than in the untreated rabbits. At 3 and 7 days, contractility of the l-NAME bladders was equal or higher than the untreated bladders. The level of hypoxia at 1 day after obstruction was significantly higher in the l-NAME-treated animals than in the untreated controls but equal at 3 and 7 days obstruction. Increased nitrotyrosine was seen by Western blot in all obstructed animals. However, the amount was significantly less in the l-NAME-treated animals at 3 and especially at 7 days. Nerve density decreased progressively after obstruction; however, it decreased to a significantly lesser degree in the l-NAME-treated bladders than in the untreated groups. These results suggest that l-NAME pretreatment enhanced ischemic damage at 1 day after obstruction but protected the bladder from nitric oxide-generated free radical damage at the later time periods by inhibiting the generation of nitrotyrosine.

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