Hierarchical data visualization of experimental erythrocyte aggregation employing cross correlation and optical flow applications.

Appraisal of microvascular erythrocyte velocity as well as aggregation are critical features of hemorheological assessment. Examination of erythrocyte velocity-aggregate characteristics is critical in assessing disorders associated with coagulopathy. Microvascular erythrocyte velocity can be assessed using various methodologic approaches; however, the shared assessment of erythrocyte velocity and aggregation has not been well described. The purpose of this study therefore is to examine three independent erythrocyte assessment strategies with and without experimentally induced aggregation in order to elucidate appropriate analytic strategy for combined velocity/aggregation assessment applicable to in-vivo capillaroscopy. We employed a hierarchical microfluidic model combined with Bland-Altman analysis to examine agreement between three methodologies to assess erythrocyte velocity appropriate for interpretation of cinematography of in-vivo microvascular hemorheology. We utilized optical and manual techniques as well as a technique which we term transversal temporal cross-correlation (TTC) to observe and measure both erythrocyte velocity and aggregation. In general, optical, manual and TTC agree in estimation of velocity at relatively low flow rate, however with an increase in infusion rate the optical flow method yielded the velocity estimates that were lower than the TTC and manual velocity estimates. We suggest that this difference was due to the fact that slower moving particles close to the channel wall were better illuminated than faster particles deeper in the channel which affected the optical flow analysis. Combined velocity/aggregation appraisal using TTC provides an efficient approach for estimating erythrocyte aggregation appropriate for in-vivo applications. We demonstrated that the optical flow and TTC analyses can be used to estimate erythrocyte velocity and aggregation both in ex-vivo microfluidics laboratory experiments as well as in-vivo recordings. The simplicity of TTC method may be advantageous for developing velocity estimate methods to be used in the clinic. The trade-off is that TTC estimation cannot capture features of the flow based on optical flow analysis of individually tracked particles.

Optogenetic Control of PIP2 Interactions Shaping ENaC Activity.

The activity of the epithelial Na+ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of ß- and γ-ENaC subunits (ßN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na+ levels [Na+]i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na+]i by measuring the fluorescent Na+ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to ßN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant ßN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na+]i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the ßN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.

Adaptive Susceptible-Infectious-Removed Model for Continuous Estimation of the COVID-19 Infection Rate and Reproduction Number in the United States: Modeling Study.

BACKGROUND: The dynamics of the COVID-19 pandemic vary owing to local population density and policy measures. During decision-making, policymakers consider an estimate of the effective reproduction number Rt, which is the expected number of secondary infections spread by a single infected individual. OBJECTIVE: We propose a simple method for estimating the time-varying infection rate and the Rt. METHODS: We used a sliding window approach with a Susceptible-Infectious-Removed (SIR) model. We estimated the infection rate from the reported cases over a 7-day window to obtain a continuous estimation of Rt. A proposed adaptive SIR (aSIR) model was applied to analyze the data at the state and county levels. RESULTS: The aSIR model showed an excellent fit for the number of reported COVID-19 cases, and the 1-day forecast mean absolute prediction error was <2.6% across all states. However, the 7-day forecast mean absolute prediction error approached 16.2% and strongly overestimated the number of cases when the Rt was rapidly decreasing. The maximal Rt displayed a wide range of 2.0 to 4.5 across all states, with the highest values for New York (4.4) and Michigan (4.5). We found that the aSIR model can rapidly adapt to an increase in the number of tests and an associated increase in the reported cases of infection. Our results also suggest that intensive testing may be an effective method of reducing Rt. CONCLUSIONS: The aSIR model provides a simple and accurate computational tool for continuous Rt estimation and evaluation of the efficacy of mitigation measures.

Gq-Coupled Muscarinic Receptor Enhancement of KCNQ2/3 Channels and Activation of TRPC Channels in Multimodal Control of Excitability in Dentate Gyrus Granule Cells.

KCNQ (Kv7, "M-type") K+ channels and TRPC (transient receptor potential, "canonical") cation channels are coupled to neuronal discharge properties and are regulated via Gq/11-protein-mediated signals. Stimulation of Gq/11-coupled receptors both consumes phosphatidylinositol 4,5-bisphosphate (PIP2) via phosphalipase Cß hydrolysis and stimulates PIP2 synthesis via rises in Ca2+i and other signals. Using brain-slice electrophysiology and Ca2+ imaging from male and female mice, we characterized threshold K+ currents in dentate gyrus granule cells (DGGCs) and CA1 pyramidal cells, the effects of Gq/11-coupled muscarinic M1 acetylcholine (M1R) stimulation on M current and on neuronal discharge properties, and elucidated the intracellular signaling mechanisms involved. We observed disparate signaling cascades between DGGCs and CA1 neurons. DGGCs displayed M1R enhancement of M-current, rather than suppression, due to stimulation of PIP2 synthesis, which was paralleled by increased PIP2-gated G-protein coupled inwardly rectifying K+ currents as well. Deficiency of KCNQ2-containing M-channels ablated the M1R-induced enhancement of M-current in DGGCs. Simultaneously, M1R stimulation in DGGCs induced robust increases in [Ca2+]i, mostly due to TRPC currents, consistent with, and contributing to, neuronal depolarization and hyperexcitability. CA1 neurons did not display such multimodal signaling, but rather M current was suppressed by M1R stimulation in these cells, similar to the previously described actions of M1R stimulation on M-current in peripheral ganglia that mostly involves PIP2 depletion. Therefore, these results point to a pleiotropic network of cholinergic signals that direct cell-type-specific, precise control of hippocampal function with strong implications for hyperexcitability and epilepsy.SIGNIFICANCE STATEMENT At the neuronal membrane, protein signaling cascades consisting of ion channels and metabotropic receptors govern the electrical properties and neurotransmission of neuronal networks. Muscarinic acetylcholine receptors are G-protein-coupled metabotropic receptors that control the excitability of neurons through regulating ion channels, intracellular Ca2+ signals, and other second-messenger cascades. We have illuminated previously unknown actions of muscarinic stimulation on the excitability of hippocampal principal neurons that include M channels, TRPC (transient receptor potential, "canonical") cation channels, and powerful regulation of lipid metabolism. Our results show that these signaling pathways, and mechanisms of excitability, are starkly distinct between peripheral ganglia and brain, and even between different principal neurons in the hippocampus.

A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels.

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

Functional responses of the hippocampus to hyperexcitability depend on directed, neuron-specific KCNQ2 K+ channel plasticity.

M-type (KCNQ2/3) K+ channels play dominant roles in regulation of active and passive neuronal discharge properties such as resting membrane potential, spike-frequency adaptation, and hyper-excitatory states. However, plasticity of M-channel expression and function in nongenetic forms of epileptogenesis are still not well understood. Using transgenic mice with an EGFP reporter to detect expression maps of KCNQ2 mRNA, we assayed hyperexcitability-induced alterations in KCNQ2 transcription across subregions of the hippocampus. Pilocarpine and pentylenetetrazol chemoconvulsant models of seizure induction were used, and brain tissue examined 48 hr later. We observed increases in KCNQ2 mRNA in CA1 and CA3 pyramidal neurons after chemoconvulsant-induced hyperexcitability at 48 hr, but no significant change was observed in dentate gyrus (DG) granule cells. Using chromogenic in situ hybridization assays, changes to KCNQ3 transcription were not detected after hyper-excitation challenge, but the results for KCNQ2 paralleled those using the KCNQ2-mRNA reporter mice. In mice 7 days after pilocarpine challenge, levels of KCNQ2 mRNA were similar in all regions to those from control mice. In brain-slice electrophysiology recordings, CA1 pyramidal neurons demonstrated increased M-current amplitudes 48 hr after hyperexcitability; however, there were no significant changes to DG granule cell M-current amplitude. Traumatic brain injury induced significantly greater KCNQ2 expression in the hippocampal hemisphere that was ipsilateral to the trauma. In vivo, after a secondary challenge with subconvulsant dose of pentylenetetrazole, control mice were susceptible to tonic-clonic seizures, whereas mice administered the M-channel opener retigabine were protected from such seizures. This study demonstrates that increased excitatory activity promotes KCNQ2 upregulation in the hippocampus in a cell-type specific manner. Such novel ion channel expressional plasticity may serve as a compensatory mechanism after a hyperexcitable event, at least in the short term. The upregulation described could be potentially leveraged in anticonvulsant enhancement of KCNQ2 channels as therapeutic target for preventing onset of epileptogenic seizures.

Intralymphatic immunotherapy for mountain cedar pollinosis: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Allergen immunotherapy can provide long-term benefits, including symptomatic relief and reduced disease progression, but it requires a lengthy regimen that presents barriers to patient adherence. Thus, there is a need for improved approaches to immunotherapy. Recently, several clinical trials have reported successful results from intralymphatic immunotherapy. OBJECTIVE: To evaluate the efficacy, safety, and tolerability of intralymphatic immunotherapy for allergies caused by mountain cedar pollen in a proof-of-concept study. METHODS: A total of 21 patients with allergic rhinoconjunctivitis because of mountain cedar pollen were randomized to receive 3 monthly intralymphatic injections of allergenic extract or placebo before the 2018-2019 mountain cedar pollen season. Safety was monitored during treatment to the end of the pollen season using structured and spontaneous reports. Clinical efficacy information was collected using a daily electronic diary of symptoms and allergy medication. Allergen-specific serum immunoglobulin E was assessed before treatment and at the end of the study. RESULTS: There were no serious adverse events or systemic reactions in either group. A total of 4 patients experienced mild injection-site reactions. Patients receiving intralymphatic immunotherapy experienced a significant improvement in allergy symptoms and medication use relative to patients receiving placebo (P < .001), and the active treatment group had lower average total combined scores on 20 of 27 days during the peak pollen season (P < .05). There was no significant difference among groups in changes to mean mountain cedar-specific serum immunoglobulin E levels. CONCLUSION: In this proof-of-concept trial, intralymphatic immunotherapy was well tolerated and improved the symptoms and medication use associated with allergic rhinoconjunctivitis caused by mountain cedar pollen. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov under the registration number NCT03682965 before the enrollment of the first subject.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains.

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

Long-term patient-reported outcomes from an open-label safety and efficacy study of bosutinib in Philadelphia chromosome-positive chronic myeloid leukemia patients resistant or intolerant to prior therapy.

BACKGROUND: Health-related quality of life (HRQOL) in patients with chronic-phase chronic myeloid leukemia (CML) is important because of the requirement for long-term treatment. This study assessed HRQOL in bosutinib-treated patients with Philadelphia chromosome-positive CML and resistance or intolerance to 1 (chronic-phase second-line [CP2L]) or more (chronic-phase third-line [CP3L]) tyrosine kinase inhibitors who had 264 weeks or more of follow-up (ClinicalTrials.gov identifier NCT00261846). METHODS: Patient-reported HRQOL was assessed with the EuroQol 5-Dimensions Questionnaire (EQ-5D) and the Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). RESULTS: In total, 284 and 119 patients composed the CP2L and CP3L cohorts, respectively. At treatment completion, more than 50% of the patients in the CP2L and CP3L cohorts completed the EQ-5D and FACT-Leu assessments. The EQ-5D and EQ-5D visual analog scale scores were stable in both cohorts throughout treatment. The mean FACT-Leu scores were generally stable over time but were lower in magnitude in the CP3L cohort versus the CP2L cohort. The FACT-Leu scale scores of a subset of patients with chronic diarrhea (CP2L, n = 101; CP3L, n = 30) were similar to the scores of the larger cohorts. Minimally important differences (MIDs) from baseline for the FACT-Leu scale scores were observed for the following: emotional well-being (EWB), Functional Assessment of Cancer Therapy-General (FACT-G) Total, FACT-Leu Total, and Functional Assessment of Cancer Therapy Trial Outcome Index (FACT-TOI) in the CP2L cohort and FACT-Leu Total in the CP3L cohort. Among patients with chronic diarrhea, MIDs were observed for EWB, FACT-G Total, FACT-Leu Total, and FACT-TOI in the CP2L cohort and for EWB, FACT-G Total, and FACT-Leu Total in the CP3L cohort. CONCLUSIONS: HRQOL was maintained with long-term bosutinib treatment for patients with CP2L and CP3L CML. Cancer 2018;124:587-95. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Safety and efficacy of second-line bosutinib for chronic phase chronic myeloid leukemia over a five-year period: final results of a phase I/II study.

Bosutinib is a Src/Abl tyrosine kinase inhibitor indicated for adults with newly-diagnosed Philadelphia positive chronic myeloid leukemia or with resistant/intolerant disease. We report the final results of a phase I/II study of second-line bosutinib in chronic phase chronic myeloid leukemia patients after imatinib failure (n=284). Median follow up and treatment durations were 54.8 (range 0.6-96.3) and 25.6 (0.2-96.3) months, respectively. At years 2 and 5, 54% and 40% of patients, respectively, remained on bosutinib. Cumulative major cytogenetic response and complete cytogenetic response rates (newly-attained or maintained from baseline) were 58% and 46%, respectively, by year 2 and 60% and 50% by year 5. Kaplan-Meier probability of maintaining major and complete cytogenetic response was 76% and 78%, respectively, at year 2 and 71% and 69% at year 5. Cumulative incidence of on-treatment disease progression/death was similar at years 5 (19%) and 2 (15%); Kaplan-Meier overall survival was 91% at year 2 and 84% at year 5. Of 169 patients who had discontinued bosutinib by year 5, 38 did so after year 2, most commonly for disease progression (n=11). Most adverse events initially occurred within two years. Overall, gastrointestinal events were the most common (diarrhea 86%, nausea 46%, vomiting 37%); the most common grade 3/4 toxicity was thrombocytopenia (25%). None of the 4 on-treatment deaths in years 3-5 were related to bosutinib. Bosutinib demonstrated durable efficacy and manageable toxicity through year 5 confirming its importance in the treatment of chronic phase chronic myeloid leukemia patients resistant/intolerant to prior imatinib. This trial was registered at clinicaltrials.gov identifier: 00261846.

Predictors of Vasopressin Responsiveness in Critically Ill Adults.

BACKGROUND: Vasopressin is commonly used as an adjunct vasopressor in shock. However, response to vasopressin varies among critically ill patients. OBJECTIVE: To identify patient-specific factors that are associated with vasopressin responsiveness in critically ill adults. METHODS: This retrospective, multicenter study included adult patients who were admitted to an intensive care unit (ICU) and received vasopressin for shock. Patients were excluded if they received vasopressin for less than 30 minutes, if vasopressin was initiated prior to ICU arrival, or if an additional vasopressor was initiated within 30 minutes of starting vasopressin. Responsiveness was defined as an increase in mean arterial pressure of ≥10 mm Hg or the ability to taper a concurrent catecholamine vasopressor. Patient-specific factors evaluated in a multivariate analysis included age, gender, ethnicity, body mass index, type of shock, serum pH, Sequential Organ Failure Assessment (SOFA) score, and use of stress-dose steroids. These variables were also evaluated in a subgroup analysis of patients with septic shock. RESULTS: Of 1619 patients screened, 400 patients were included, with 231 identified as vasopressin responsive and 169 as nonresponsive. Vasopressin used as an adjunct vasopressor, as opposed to first line, during shock was the only variable associated with vasopressin responsiveness (odds ratio [OR] = 1.71; 95% CI = 1.10 to 2.65). Among the subgroup of patients with septic shock, female patients had a higher odds of responding than male patients (OR = 2.10; 95% CI = 1.12 to 3.95). CONCLUSIONS: Vasopressin initiated as an adjunct vasopressor, as opposed to first-line therapy, was associated with response.

CT Findings of Traumatic Bucket-Handle Mesenteric Injuries.

OBJECTIVE: The purpose of this study is to describe a specific type of blunt traumatic mesenteric injury called a bucket-handle tear, review its varied CT appearances, and discuss the role of CT in its detection. CONCLUSION: A bucket-handle mesenteric injury is a rare but life-threatening blunt traumatic injury that can be difficult to detect prospectively on CT and for which delays in diagnosis and definitive surgical management can result in poor outcomes.

Analgesia and Sedation Requirements in Mechanically Ventilated Trauma Patients With Acute, Preinjury Use of Cocaine and/or Amphetamines.

BACKGROUND: The purpose of this study was to determine whether mechanically ventilated trauma patients with a positive urine drug screen (UDS) for cocaine and/or amphetamines have different opioid analgesic and sedative requirements compared with similar patients with a negative drug screen for these stimulants. METHODS: This retrospective, single-center cohort study at a tertiary care, academic medical and level 1 trauma center in the United States included patients ≥16 years of age who were admitted to an adult intensive care unit with a diagnosis of trauma between 2009 and 2013 with a UDS documented within 24 hours of admission, and were mechanically ventilated for >24 hours. The primary end point was the daily dose of opioid received during mechanical ventilation, expressed as morphine equivalents, for patients presenting with a positive UDS for cocaine and/or amphetamines compared with patients with a negative UDS for these stimulants. Secondary end points included the daily benzodiazepine dose and median infusion rates of propofol and dexmedetomidine received during mechanical ventilation, duration of mechanical ventilation, intensive care unit and hospital length of stay, and in-hospital mortality. Analgesic and sedative goals were similar for the duration of the study period, and both intermittent and continuous infusions of opioids and sedatives were administered to achieve these targets, although a standardized approach was not used. A multivariate logistic regression analysis and a propensity-adjusted model evaluated patient characteristics predictive of a higher median opioid requirement. RESULTS: A total of 150 patients were included in the final analysis. In a univariate analysis, opioid and sedative requirements were similar for patients presenting with a positive UDS for cocaine and/or amphetamines compared with patients with a negative UDS for these stimulants. In the multivariate regression analysis, increasing age and Abbreviated Injury Scale (head and neck) were associated with decreased daily opioid requirements (odds ratio [OR], .95, 95% confidence interval [CI], .93-.97 and OR, .71, 95% CI, .65-.77, respectively), whereas preinjury stimulant use was not predictive of opioid requirements (OR, .88, 95% CI, .40-1.90). In a propensity score--adjusted model, preinjury stimulant use was similarly not predictive of opioid requirements during mechanical ventilation (OR, .97, 95% CI, .44-2.11). CONCLUSIONS: For trauma patients presenting with acute, preinjury use of cocaine and/or amphetamines, analgesic and sedative requirements are variables and may not be greater than those patients presenting with a stimulant-negative UDS to achieve desirable pain control and depth of sedation, although this observation should be interpreted cautiously in light of the wide CI observed in the propensity score--adjusted model. Although unexpected, these findings indicate that empirically increasing analgesic and sedative doses based on positive UDS results for these stimulants may not be necessary.

Augmentation of M-type (KCNQ) potassium channels as a novel strategy to reduce stroke-induced brain injury.

Cerebral ischemic stroke is a worldwide cause of mortality/morbidity and thus an important focus of research to decrease the severity of brain injury. Therapeutic options for acute stroke are still limited. In neurons throughout the brain, "M-type" K(+) currents, underlain by KCNQ subunits 2-5, play dominant roles in control over excitability, and are thus implicated in myriad neurological and psychiatric disorders. Although KCNQ channel openers, such as retigabine, have emerged as anti-epilepsy drugs, their effects on ischemic injury remain unknown. Here, we investigated the protective effects of M-channel openers on stroke-induced brain injury in mouse photothrombotic and middle cerebral artery occlusion (MCAo) models. Both photothrombosis and MCAo led to rapid, predictable, and consistently sized necrotic brain lesions, inflammatory responses, and behavioral deficits. Administration of three distinct M-channel openers at 0-6 h after ischemic injury significantly decreased brain infarct size and inflammation, and prevented neurological dysfunction, although they were more effective when administered 0-3 h poststroke. Thus, we show beneficial effects against stroke-induced brain injury and neuronal death through pharmacological regulation of ion channels that control neuronal excitability.

Mechanisms and dynamics of AKAP79/150-orchestrated multi-protein signalling complexes in brain and peripheral nerve.

A-kinase anchoring proteins (AKAPs) have emerged as a converging point of diverse signals to achieve spatiotemporal resolution of directed cellular regulation. With the extensive studies of AKAP79/150 in regulation of ion channel activity, the major questions to be posed centre on the mechanism and functional role of synergistic regulation of ion channels by such signalling proteins. In this review, we summarize recent discoveries of AKAP79/150-mediated modulation of voltage-gated neuronal M-type (KCNQ, Kv7) K(+) channels and L-type CaV 1 Ca(2+) channels, on both short- and longer-term time scales, highlighting the dynamics of the macromolecular signalling complexes in brain and peripheral nerve We also discuss several models for the possible mechanisms of these multi-protein assemblies and how they serve the agenda of the neurons in which they occur.

Factors influencing long-term efficacy and tolerability of bosutinib in chronic phase chronic myeloid leukaemia resistant or intolerant to imatinib.

The dual SRC/ABL1 tyrosine kinase inhibitor bosutinib is indicated for adults with Ph+ chronic myeloid leukaemia (CML) resistant/intolerant to prior therapy. This analysis of an ongoing phase 1/2 study (NCT00261846) assessed effects of baseline patient characteristics on long-term efficacy and safety of bosutinib 500 mg/day in adults with imatinib (IM)-resistant (IM-R; n = 196)/IM-intolerant (IM-I; n = 90) chronic phase (CP) CML. Median treatment duration was 24·8 months (median follow-up, 43·6 months). Cumulative major cytogenetic response (MCyR) rate [95% confidence interval (CI)], was 59% (53-65%); Kaplan-Meier (KM) probability of maintaining MCyR at 4 years was 75% (66-81%). Cumulative incidence of on-treatment progression/death at 4 years was 19% (95% CI, 15-24%); KM 2-year overall survival was 91% (87-94%). Significant baseline predictors of both MCyR and complete cytogenetic response (newly attained/maintained from baseline) at 3 and 6 months included prior IM cytogenetic response, baseline MCyR, prior interferon therapy and <6 months duration from diagnosis to IM treatment initiation and no interferon treatment before IM. The most common adverse event (AE) was diarrhoea (86%). Baseline bosutinib-sensitive BCR-ABL1 mutation was the only significant predictor of grade 3/4 diarrhoea; no significant predictors were identified for liver-related AEs. Bosutinib demonstrates durable efficacy and manageable toxicity in IM-R/IM-I CP-CML patients.

Long-term evaluation of cardiac and vascular toxicity in patients with Philadelphia chromosome-positive leukemias treated with bosutinib.

Vascular and cardiac safety during tyrosine kinase inhibitor (TKI) therapy is an emerging issue. We evaluated vascular/cardiac toxicities associated with long-term bosutinib treatment for Philadelphia chromosome-positive (Ph+) leukemia based on treatment-emergent adverse events (TEAEs) and changes in QTc intervals and ejection fraction in two studies: a phase 1/2 study of second-/third-/fourth-line bosutinib for Ph+ leukemia resistant/intolerant to prior TKIs (N = 570) and a phase 3 study of first-line bosutinib (n = 248) versus imatinib (n = 251) in chronic phase chronic myeloid leukemia. Follow-up time was ≥48 months (both studies). Incidences of vascular/cardiac TEAEs in bosutinib-treated patients were 7%/10% overall with similar incidences observed with first-line bosutinib (5%/8%) and imatinib (4%/6%). Few patients had grade ≥3 vascular/cardiac events (4%/4%) and no individual TEAE occurred in >2% of bosutinib patients. Exposure-adjusted vascular/cardiac TEAE rates (patients with events/patient-year) were low for second-line or later bosutinib (0.037/0.050) and not significantly different between first-line bosutinib (0.015/0.024) and imatinib (0.011/0.017; P ≥ 0.267). Vascular/cardiac events were managed mainly with concomitant medications (39%/44%), bosutinib treatment interruptions (18%/21%), or dose reductions (4%/8%); discontinuations due to these events were rare (0.7%/1.0%). Based on logistic regression modelling, performance status >0 and history of vascular or cardiac disorders were prognostic of vascular/cardiac events in relapsed/refractory patients; hyperlipidemia/hypercholesterolemia and older age were prognostic of cardiac events. In newly diagnosed patients, older age was prognostic of vascular/cardiac events; history of diabetes was prognostic of vascular events. Incidences of vascular and cardiac events were low with bosutinib in the first-line and relapsed/refractory settings following long-term treatment in patients with Ph+ leukemia. Am. J. Hematol. 91:606-616, 2016. © 2016 Wiley Periodicals, Inc.

Long-term efficacy and safety of bosutinib in patients with advanced leukemia following resistance/intolerance to imatinib and other tyrosine kinase inhibitors.

Long-term efficacy and safety of bosutinib (≥4 years follow-up from last enrolled patient) were evaluated in an ongoing phase 1/2 study in the advanced leukemia cohort with prior treatment failure (accelerated-phase [AP, n = 79] chronic myeloid leukemia [CML], blast-phase [BP, n = 64] CML, acute lymphoblastic leukemia [ALL, n = 24]). Fourteen AP, 2 BP, and 1 ALL patient remained on bosutinib at 4 years (vs. 38, 8, 1 at 1 year); median (range) treatment durations: 10.2 (0.1-88.6), 2.8 (0.03-55.9), 0.97 (0.3-89.2) months. Among AP and BP patients, 57% and 28% newly attained or maintained baseline overall hematologic response (OHR); 40% and 37% attained/maintained major cytogenetic response (MCyR) by 4 years (most by 12 months). In responders at 1 versus 4 years, Kaplan-Meier (KM) probabilities of maintaining OHR were 78% versus 49% (AP) and 28% versus 19% (BP); KM probabilities of maintaining MCyR were 65% versus 49% (AP) and 21% versus 21% (BP). Most common AEs (AP, BP) were gastrointestinal (96%; 83%), primarily diarrhea (85%; 64%), which was typically low grade (maximum grade 1/2: 81%; 59%) and transient; no patient discontinued due to diarrhea. Serious AEs occurred in 44 (56%) AP and 37 (58%) BP patients, most commonly pneumonia (n = 9) for AP and pyrexia (n = 6) for BP; 11 and 13 died within 30 days of last dose (2 considered bosutinib-related [AP] per investigator). Responses were durable in ∼50% AP responders at 4 years (∼25% BP patients responded at year 1, suggesting possible bridge-to-transplant role in BP patients); toxicity was manageable.