CAPILLARY LEAK AND EDEMA AFTER RESUSCITATION: THE POTENTIAL CONTRIBUTION OF REDUCED ENDOTHELIAL SHEAR STRESS CAUSED BY HEMODILUTION.

ABSTRACT: Normal shear stress is essential for the normal structure and functions of the microcirculation. Hemorrhagic shock leads to reduced shear stress due to reduced tissue perfusion. Although essential for the urgent restoration of cardiac output and systemic blood pressure, large volume resuscitation with currently available solutions causes hemodilution, further reducing endothelial shear stress. In this narrative review, we consider how the use of currently available resuscitation solutions results in persistent reduction in endothelial shear stress, despite successfully increasing cardiac output and systemic blood pressure. We consider how this reduced shear stress causes (1) a failure to restore normal vasomotor function and normal tissue perfusion thus leading to persistent tissue hypoxia and (2) increased microvascular endothelial permeability resulting in edema formation and impaired organ function. We discuss the need for clinical research into resuscitation strategies and solutions that aim to quickly restore endothelial shear stress in the microcirculation to normal.

Early warning scores and critical care transfer - patient heterogeneity, low sensitivity, high mortality.

BACKGROUND: Emergency warning systems (EWS) are becoming a standard of care, but have unproven screening value in early critical illness. Similarly, emergency response team (ERT) care is of uncertain value. These questions are most controversial in mixed patient populations, where screening performance might vary, and intensivist-led ERT care might divert resources from existing patients. AIMS: To examine triggering events, disposition and outcome data for an intensivist-staffed EWS-ERT system. METHODS: We analysed process and outcome data over three years, classing EWS-triggered patients into three categories (non-escalated, escalated ward care and critical care transfer). The relationships between EWS data, pre-triggering clinical data, and patient disposition and outcome were examined. RESULTS: There were 1675 calls in 1190 patients. Most occurred later during admission, with critical care transfer in a minority; the rest were followed by escalated or non-escalated ward care. Patients transferred to critical care had high mortality (40.3%); less than half of patient transfers occurred following triggering EWS score predicted overall hospital mortality, but not mortality after critical care. CONCLUSIONS: In a diverse hospital population, most triggering patients did not receive critical care and most critical care transfers occurred without triggering. Triggering was an insensitive screening measure for critical illness, followed by poor outcome. Higher scores predicted higher probability of transfer, but not later mortality, suggesting that EWS is being used as a decision aid but is not a true severity of illness score. Other, non-EWS data are needed for earlier detection and for prioritizing access to critical care.

Structure-Guided Optimization Provides a Series of TTK Protein Inhibitors with Potent Antitumor Activity.

TTK is an essential spindle assembly checkpoint enzyme in many organisms. It plays a central role in tumor cell proliferation and is aberrantly overexpressed in a wide range of tumor types. We recently reported on a series of potent and selective TTK inhibitors with strong antiproliferative activity in triple negative breast cancer (TNBC) cell lines (8: TTK IC50 = 3.0 nM; CAL-51 IC50 = 84.0 nM). Inspired by previously described potent tricyclic TTK inhibitor 6 (TTK IC50 = 0.9 nM), we embarked on a structure-enabled design and optimization campaign to identify an improved series with excellent potency, TTK selectivity, solubility, CYP inhibition profile, and in vivo efficacy in a TNBC xenograft model. These efforts culminated in the discovery of 25 (TTK IC50 = 3.0 nM; CAL-51 IC50 = 16.0 nM), which showed significant single-agent efficacy when dosed iv in a TNBC xenograft model without body weight loss.

Erector Spinae Plane Blocks in Major Hepatopancreaticobiliary Surgery: A Case Series.

Hepatopancreaticobiliary (HPB) surgery is major upper abdominal surgery with considerable risk of pulmonary complications related to postoperative pain. While epidural analgesia remains an effective analgesic technique for upper abdominal surgery, HPB surgery poses challenges to its use due to coagulopathy. Erector spinae plane (ESP) blocks are a promising alternative to epidurals. Injection of local anesthetic deep to the erector spinae muscle plane and placement of a catheter for prolonged effect provide both somatic and visceral analgesia for both thoracic and abdominal surgery. We describe a series of 3 cases that illustrate the efficacy of ESP blocks after major HPB surgery.

Design and Optimization Leading to an Orally Active TTK Protein Kinase Inhibitor with Robust Single Agent Efficacy.

Triple negative breast cancer (TNBC) is an aggressive disease with high relapse rates and few treatment options. Outlined in previous publications, we identified a series of potent, dual TTK/CLK2 inhibitors with strong efficacy in TNBC xenograft models. Pharmacokinetic properties and kinome selectivity were optimized, resulting in the identification of a new series of potent, selective, and orally bioavailable TTK inhibitors. We describe here the structure-activity relationship of the 2,4-disubstituted-7 H-pyrrolo[2,3- d]pyrimidine series, leading to significant single agent efficacy in a TNBC xenograft model without body weight loss. The design effort evolving an iv-dosed TTK/CLK2 inhibitor to an orally bioavailable TTK inhibitor is described.

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1-S Checkpoint.

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1-S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1-S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1-S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727-38. ©2018 AACR.

The NAPRESSIM trial: the use of low-dose, prophylactic naloxone infusion to prevent respiratory depression with intrathecally administered morphine in elective hepatobiliary surgery: a study protocol and statistical analysis plan for a randomised controlled trial.

BACKGROUND: Intrathecally administered morphine is effective as part of a postoperative analgesia regimen following major hepatopancreaticobiliary surgery. However, the potential for postoperative respiratory depression at the doses required for effective analgesia currently limits its clinical use. The use of a low-dose, prophylactic naloxone infusion following intrathecally administered morphine may significantly reduce postoperative respiratory depression. The NAPRESSIM trial aims to answer this question. METHODS/DESIGN: 'The use of low-dose, prophylactic naloxone infusion to prevent respiratory depression with intrathecally administered morphine' trial is an investigator-led, single-centre, randomised, double-blind, placebo-controlled, double-arm comparator study. The trial will recruit 96 patients aged > 18 years, undergoing major open hepatopancreaticobiliary resections, who are receiving intrathecally administered morphine as part of a standard anaesthetic regimen. It aims to investigate whether the prophylactic administration of naloxone via intravenous infusion compared to placebo will reduce the proportion of episodes of respiratory depression in this cohort of patients. Trial patients will receive an infusion of naloxone or placebo, commenced within 1 h of postoperative extubation continued until the first postoperative morning. The primary outcome is the rate of respiratory depression in the intervention group as compared to the placebo group. Secondary outcomes include pain scores, rates of nausea and vomiting, pruritus, sedation scores and adverse outcomes. We will also employ a novel, non-invasive, respiratory minute volume monitor (ExSpiron 1Xi, Respiratory Motion, Inc., 411 Waverley Oaks Road, Building 1, Suite 150, Waltham, MA, USA) to assess the monitor's accuracy for detecting respiratory depression. DISCUSSION: The trial aims to provide a clear management plan to prevent respiratory depression after the intrathecal administration of morphine, and thereby improve patient safety. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02885948 . Registered retrospectively on 4 July 2016. Protocol Version 2.0, 3 April 2017. Protocol identification (code or reference number): UCDCRC/15/006 EudraCT registration number: 2015-003504-22. Registered on 5 August 2015.

The Discovery of a Dual TTK Protein Kinase/CDC2-Like Kinase (CLK2) Inhibitor for the Treatment of Triple Negative Breast Cancer Initiated from a Phenotypic Screen.

Triple negative breast cancer (TNBC) remains a serious unmet medical need with discouragingly high relapse rates. We report here the synthesis and structure-activity relationship (SAR) of a novel series of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with potent activity against TNBC tumor cell lines. These compounds were discovered from a TNBC phenotypic screen and possess a unique dual inhibition profile targeting TTK (mitotic exit) and CLK2 (mRNA splicing). Design and optimization, driven with a TNBC tumor cell assay, identified potent and selective compounds with favorable in vitro and in vivo activity profiles and good iv PK properties. This cell-based driven SAR produced compounds with strong single agent in vivo efficacy in multiple TNBC xenograft models without significant body weight loss. These data supported the nomination of CC-671 into IND-enabling studies as a single agent TNBC therapy.

Prolonged Unexplained Hypoxemia as Initial Presentation of Cirrhosis: A Case Report.

BACKGROUND Hepatopulmonary syndrome (HPS) is a pulmonary complication of advanced liver disease with dyspnea as the predominant presenting symptom. The diagnosis of HPS can often be missed due to its nonspecific presentation and the presence of other comorbidities. CASE REPORT We present an interesting case of an obese 43-year-old man who presented with progressive, unexplained hypoxemia and shortness of breath in the absence of any symptoms or signs of chronic liver disease. After extensive cardiopulmonary investigations, he was diagnosed with severe HPS as a result of non-alcoholic steatohepatitis (NASH) leading to cirrhosis. He subsequently underwent successful hepatic transplantation and continues to improve at 12-month follow-up. CONCLUSIONS HPS needs to be considered in the differential diagnosis of unexplained hypoxemia. Given its poor prognosis, early diagnosis is warranted and treatment with liver transplantation is the preferred choice.

Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor ß1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor ß stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

Systematic Reviews of Anesthesiologic Interventions Reported as Statistically Significant: Problems with Power, Precision, and Type 1 Error Protection.

BACKGROUND: The GRADE Working Group assessment of the quality of evidence is being used increasingly to inform clinical decisions and guidelines. The assessment involves explicit consideration of all sources of uncertainty. One of these sources is imprecision or random error. Many published meta-analyses are underpowered and likely to be updated in the future. When data are sparse and there are repeated updates, the risk of random error is increased. Trial Sequential Analysis (TSA) is one of several methodologies that estimates this increased risk (and decreased precision) in meta-analyses. With nominally statistically significant meta-analyses of anesthesiologic interventions, we used TSA to estimate power and imprecision in the context of sparse data and repeated updates. METHODS: We conducted a search to identify all systematic reviews with meta-analyses that investigated an intervention that may be implemented by an anesthesiologist during the perioperative period. We randomly selected 50 meta-analyses that reported a statistically significant dichotomous outcome in their abstract. We applied TSA to these meta-analyses by using 2 main TSA approaches: relative risk reduction 20% and relative risk reduction consistent with the conventional 95% confidence limit closest to null. We calculated the power achieved by each included meta-analysis, by using each TSA approach, and we calculated the proportion that maintained statistical significance when allowing for sparse data and repeated updates. RESULTS: From 11,870 titles, we found 682 systematic reviews that investigated anesthesiologic interventions. In the 50 sampled meta-analyses, the median number of trials included was 8 (interquartile range [IQR], 5-14), the median number of participants was 964 (IQR, 523-1736), and the median number of participants with the outcome was 202 (IQR, 96-443). By using both of our main TSA approaches, only 12% (95% CI, 5%-25%) of the meta-analyses had power ≥ 80%, and only 32% (95% CI, 20%-47%) of the meta-analyses preserved the risk of type 1 error <5%. CONCLUSIONS: Most nominally statistically significant meta-analyses of anesthesiologic interventions are underpowered, and many do not maintain their risk of type 1 error <5% if TSA monitoring boundaries are applied. Consideration of the effect of sparse data and repeated updates is needed when assessing the imprecision of meta-analyses of anesthesiologic interventions.

Inhibition of the NOTCH pathway using γ-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors.

Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

Remote ischemic preconditioning does not affect the incidence of acute kidney injury after elective abdominal aortic aneurysm repair.

OBJECTIVE: Open abdominal aortic aneurysm (AAA) repair is associated with a high risk of renal injury with few known strategies demonstrating a reduction in this risk. Remote ischemic preconditioning (RIPC) has been identified as having the potential to minimize organ injury following major vascular surgery. This trial investigated the potential for RIPC to attenuate renal and myocardial injury in patients undergoing elective open AAA repair. DESIGN: Prospective, randomized double-blinded control trial. SETTING: Tertiary referral hospital. PARTICIPANTS: Sixty-two patients undergoing elective open AAA repair. INTERVENTION: RIPC was achieved via three 5-minute cycles of upper limb ischemia using a blood pressure cuff or control (sham cuff). MEASUREMENTS: Primary outcome was the occurrence of renal injury, as measured by an increase in creatinine during the first 4 postoperative days. Secondary outcomes included urinary neutrophil-gelatinase-associated lipocalin (NGAL), occurrence of acute kidney injury (AKI), occurrence of myocardial injury as defined by troponin rise, incidence of postoperative complications, and mortality. There was no difference in postoperative creatinine levels, NGAL levels, or the occurrence of AKI between the groups at any postoperative time point. Similarly, there was no difference in the occurrence of myocardial injury or mortality. Of note, 6 patients in the RIPC group, while no patient in the control group, experienced postoperative complications that required repeat surgical laparotomy, potentially masking any renoprotective effects of RIPC. CONCLUSION: RIPC did not reduce the risk of postoperative renal failure or myocardial injury in patients undergoing open AAA repair. The authors' results do not support the introduction of this technique to routine clinical practice.

Discovery of siRNA lipid nanoparticles to transfect suspension leukemia cells and provide in vivo delivery capability.

As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

Acute respiratory distress syndrome: underrecognition by clinicians.

BACKGROUND: Previous reports suggest that acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is underdiagnosed in both adult and pediatric clinical practice. Underrecognition of this condition may be a barrier to instituting a low tidal volume ventilation strategy. This study aimed to determine the accuracy of clinical diagnoses of ARDS in daily practice using the American European Consensus Conference (AECC) criteria as a criterion standard and to investigate whether clinical recognition of ARDS altered ventilator management. METHODS: This retrospective study included intensive care unit (ICU) patients who died and underwent postmortem examination. Two independent reviewers assigned each patient to those with ALI/ARDS or no ALI. For those who met AECC criteria for ARDS, all patient records were reviewed for the presence of a documented diagnosis of the condition. The accuracy of the clinicians in diagnosing ALI/ARDS was determined, and ventilator settings between the clinically "diagnosed" and "non-diagnosed" groups were compared. The diagnostic accuracy in predetermined subgroups (those with diffuse alveolar damage, with ≥3 affected chest x-ray quadrants, with diagnosis≥3 days, with pulmonary vs extrapulmonary cause) was also examined. RESULTS: Of 98 consecutive ICU patients who died and underwent autopsy, 51 met the inclusion criteria. Sixteen of 51 patients (31.3%) who had ALI/ARDS according to the AECC criteria had this recorded in their clinical notes. Those with histologic evidence of ALI/ARDS (diffuse alveolar damage) and with a more severe chest x-ray pattern or who satisfied the criteria for a number of consecutive days were no more likely to have a clinical diagnosis of ALI/ARDS recorded. However, those with a pulmonary cause of ALI/ARDS were more likely to have a diagnosis recorded. Tidal volumes, positive end-expiratory pressure, and mean airway pressure were higher in those with a clinical diagnosis of ARDS. CONCLUSIONS: Acute respiratory distress syndrome is underrecognized by clinicians in ICU, and recognition does not result in lower tidal volume ventilation. Significant barriers remain to the recognition of ALI/ARDS and application of an evidence-based ventilator strategy.

Hypoxia-induced inflammation in the lung: a potential therapeutic target in acute lung injury?

Acute lung injury (ALI) is a severe form of hypoxic lung disease responsible for a large number of deaths worldwide. Despite recent advances in supportive care, no reduction in mortality has been evident since the introduction of a standard consensus definition almost two decades ago. New strategies are urgently required to help design effective therapies for this condition. A key pathological feature of ALI involves regional alveolar hypoxia. Because alveolar hypoxia in isolation, such as that encountered at high altitude, causes an inflammatory pulmonary phenotype in the absence of any other pathogenic stimuli, these regions may not be passive bystanders but may actually contribute to the pathogenesis and progression of lung injury. Unique transcriptional responses to hypoxia in the lung apparently allow it to express an inflammatory phenotype at levels of hypoxia that would not produce such a response in other organs. We will review recent advances in our understanding of these unique transcriptional responses to moderate levels of alveolar hypoxia, which may provide new insights into the pathogenesis of ALI.

BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.