Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells.

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese hamster ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with nine different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

Ethical challenges and moral distress among field epidemiologists.

BACKGROUND: As 'disease detectives' and directors of public health programs, field epidemiologists play essential roles in protecting public health. Although ethical issues receive considerable attention in medical and research settings, less is known about ethical challenges faced by field epidemiologists in public health programs. Similarly, little is known about moral distress among field epidemiologists, i.e., situations in which they are constrained from acting on what they know to be morally right. Moral distress is strongly associated with empathy fatigue, burnout, reduced job retention, and disengagement. To better understand ethics training needs for field epidemiologists, in February 2019, members of TEPHIConnect, an online and mobile networking platform for Field Epidemiology Training Program (FETP) alumni, were invited to participate in an anonymous survey about ethical challenges and moral distress. RESULTS: Among 126 respondents from 54 countries, leading causes of ethical dilemmas included inadequate informed consent (61%), inequitable allocation of resources (49%), and conflicts of interest (43%). These occur primarily in settings of disease outbreaks (60%); research (55%); and public health programs at the state, province, or national level (45%) or community level (43%). Work-related moral distress was reported by 91% of respondents, including 26% who experience it "frequently" or "almost always." Field epidemiologists working in low- and low-middle income countries were more likely to report moral distress "frequently" or "almost always" than those in higher-income countries (33.0% vs 9.1%, P = 0.006). The most common perceived contributors to moral distress included excessive stress and work demands (30%) and inadequate support from leaders (25%). CONCLUSIONS: Field epidemiologists face significant work-related ethical challenges, which are endemic to public health and political systems. A substantial proportion of field epidemiologists also experience some degree of moral distress, often in association with these challenges. These findings indicate an unmet need among field epidemiologists for support in navigating ethical challenges, as well as for resources to address the human and professional consequences of moral distress.

Thrombolytic removal of intraventricular haemorrhage in treatment of severe stroke: results of the randomised, multicentre, multiregion, placebo-controlled CLEAR III trial.

BACKGROUND: Intraventricular haemorrhage is a subtype of intracerebral haemorrhage, with 50% mortality and serious disability for survivors. We aimed to test whether attempting to remove intraventricular haemorrhage with alteplase versus saline irrigation improved functional outcome. METHODS: In this randomised, double-blinded, placebo-controlled, multiregional trial (CLEAR III), participants with a routinely placed extraventricular drain, in the intensive care unit with stable, non-traumatic intracerebral haemorrhage volume less than 30 mL, intraventricular haemorrhage obstructing the 3rd or 4th ventricles, and no underlying pathology were adaptively randomly assigned (1:1), via a web-based system to receive up to 12 doses, 8 h apart of 1 mg of alteplase or 0·9% saline via the extraventricular drain. The treating physician, clinical research staff, and participants were masked to treatment assignment. CT scans were obtained every 24 h throughout dosing. The primary efficacy outcome was good functional outcome, defined as a modified Rankin Scale score (mRS) of 3 or less at 180 days per central adjudication by blinded evaluators. This study is registered with ClinicalTrials.gov, NCT00784134. FINDINGS: Between Sept 18, 2009, and Jan 13, 2015, 500 patients were randomised: 249 to the alteplase group and 251 to the saline group. 180-day follow-up data were available for analysis from 246 of 249 participants in the alteplase group and 245 of 251 participants in the placebo group. The primary efficacy outcome was similar in each group (good outcome in alteplase group 48% vs saline 45%; risk ratio [RR] 1·06 [95% CI 0·88-1·28; p=0·554]). A difference of 3·5% (RR 1·08 [95% CI 0·90-1·29], p=0·420) was found after adjustment for intraventricular haemorrhage size and thalamic intracerebral haemorrhage. At 180 days, the treatment group had lower case fatality (46 [18%] vs saline 73 [29%], hazard ratio 0·60 [95% CI 0·41-0·86], p=0·006), but a greater proportion with mRS 5 (42 [17%] vs 21 [9%]; RR 1·99 [95% CI 1·22-3·26], p=0·007). Ventriculitis (17 [7%] alteplase vs 31 [12%] saline; RR 0·55 [95% CI 0·31-0·97], p=0·048) and serious adverse events (114 [46%] alteplase vs 151 [60%] saline; RR 0·76 [95% CI 0·64-0·90], p=0·002) were less frequent with alteplase treatment. Symptomatic bleeding (six [2%] in the alteplase group vs five [2%] in the saline group; RR 1·21 [95% CI 0·37-3·91], p=0·771) was similar. INTERPRETATION: In patients with intraventricular haemorrhage and a routine extraventricular drain, irrigation with alteplase did not substantially improve functional outcomes at the mRS 3 cutoff compared with irrigation with saline. Protocol-based use of alteplase with extraventricular drain seems safe. Future investigation is needed to determine whether a greater frequency of complete intraventricular haemorrhage removal via alteplase produces gains in functional status. FUNDING: National Institute of Neurological Disorders and Stroke.

Accuracy of the ABC/2 Score for Intracerebral Hemorrhage: Systematic Review and Analysis of MISTIE, CLEAR-IVH, and CLEAR III.

BACKGROUND AND PURPOSE: The ABC/2 score estimates intracerebral hemorrhage (ICH) volume, yet validations have been limited by small samples and inappropriate outcome measures. We determined accuracy of the ABC/2 score calculated at a specialized reading center (RC-ABC) or local site (site-ABC) versus the reference-standard computed tomography-based planimetry (CTP). METHODS: In Minimally Invasive Surgery Plus Recombinant Tissue-Type Plasminogen Activator for Intracerebral Hemorrhage Evacuation-II (MISTIE-II), Clot Lysis Evaluation of Accelerated Resolution of Intraventricular Hemorrhage (CLEAR-IVH) and CLEAR-III trials. ICH volume was prospectively calculated by CTP, RC-ABC, and site-ABC. Agreement between CTP and ABC/2 was defined as an absolute difference up to 5 mL and relative difference within 20%. Determinants of ABC/2 accuracy were assessed by logistic regression. RESULTS: In 4369 scans from 507 patients, CTP was more strongly correlated with RC-ABC (r(2)=0.93) than with site-ABC (r(2)=0.87). Although RC-ABC overestimated CTP-based volume on average (RC-ABC, 15.2 cm(3); CTP, 12.7 cm3), agreement was reasonable when categorized into mild, moderate, and severe ICH (κ=0.75; P<0.001). This was consistent with overestimation of ICH volume in 6 of 8 previous studies. Agreement with CTP was greater for RC-ABC (84% within 5 mL; 48% of scans within 20%) than for site-ABC (81% within 5 mL; 41% within 20%). RC-ABC had moderate accuracy for detecting ≥5 mL change in CTP volume between consecutive scans (sensitivity, 0.76; specificity, 0.86) and was more accurate with smaller ICH, thalamic hemorrhage, and homogeneous clots. CONCLUSIONS: ABC/2 scores at local or central sites are sufficiently accurate to categorize ICH volume and assess eligibility for the CLEAR-III and MISTIE III studies and moderately accurate for change in ICH volume. However, accuracy decreases with large, irregular, or lobar clots. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: MISTIE-II NCT00224770; CLEAR-III NCT00784134.

Falcine and Tentorial Subdural Hematomas May Not Routinely Require Transfer to a Tertiary Care Center.

BACKGROUND: Patients with subdural hematomas (SDH) are frequently transferred to tertiary care centers. Although many prognostic factors, treatment strategies, and outcomes for convexity SDH have been reported, little is known about falcine and tentorial SDH. OBJECTIVES: To describe features and outcomes of isolated falcine and tentorial SDH. METHODS: We reviewed clinical/radiographic findings, treatment, length of stay (LOS), and outcome of adult patients transferred to a tertiary care center for acute SDH. Characteristics of patients with isolated falcine/tentorial SDH and outcomes (favorable [discharge to home/acute rehabilitation] vs. unfavorable [death/hospice/skilled nursing facility/long term care]) were assessed with univariate analyses. RESULTS: Of 210 patients with SDH, mean age was 69.5 years; 117 were male; 98 (47%) underwent surgical SDH evacuation. Twenty-seven patients had isolated falcine or tentorial SDH, with known traumatic etiology in 23. None of the falcine/tentorial SDH patients required surgery or intubation. Compared with convexity SDH, patients with falcine/tentorial SDH were younger (59.7 vs. 70.9 years, p = 0.01), had higher admission Glasgow Coma Scale scores at the referring (p = 0.01) and receiving facility (p = 0.004), and shorter median intensive care unit LOS (1 vs. 3, p < 0.0001). All patients (100%) with falcine/tentorial SDH had favorable outcome vs. 68% with convexity SDH (p = 0.0005). CONCLUSION: Isolated tentorial/falcine SDH without associated neurological deficits represent a benign entity among acute SDH, with no need for surgical intervention, short LOS, and favorable outcome. Our data indicate that for these patients, in the absence of complicating factors, transfer to a tertiary care center may not be routinely indicated.

Optimizing prediction scores for poor outcome after intra-arterial therapy in anterior circulation acute ischemic stroke.

BACKGROUND AND PURPOSE: Intra-arterial therapy (IAT) promotes recanalization of large artery occlusions in acute ischemic stroke. Despite high recanalization rates, poor clinical outcomes are common. We attempted to optimize a score that combines clinical and imaging variables to more accurately predict poor outcome after IAT in anterior circulation occlusions. METHODS: Patients with acute ischemic stroke undergoing IAT at University of Texas (UT) Houston for large artery occlusions (middle cerebral artery or internal carotid artery) were reviewed. Independent predictors of poor outcome (modified Rankin Scale, 4-6) were studied. External validation was performed on IAT-treated patients at Emory University. RESULTS: A total of 163 patients were identified at UT Houston. Independent predictors of poor outcome (P≤0.2) were identified as score variables using sensitivity analysis and logistic regression. Houston Intra-Arterial Therapy 2 (HIAT2) score ranges 0 to 10: age (≤59=0, 60-79=2, ≥80 years=4), glucose (<150=0, ≥150=1), National Institute Health Stroke Scale (≤10=0, 11-20=1, ≥21=2), the Alberta Stroke Program Early CT Score (8-10=0, ≤7=3). Patients with HIAT2≥5 were more likely to have poor outcomes at discharge (odds ratio, 6.43; 95% confidence interval, 2.75-15.02; P<0.001). After adjusting for reperfusion (Thrombolysis in Cerebral Infarction score≥2b) and time from symptom onset to recanalization, HIAT2≥5 remained an independent predictor of poor outcome (odds ratio, 5.88; 95% confidence interval, 1.96-17.64; P=0.02). Results from the cohort of Emory (198 patients) were consistent; patients with HIAT2 score≥5 had 6× greater odds of poor outcome at discharge and at 90 days. HIAT2 outperformed other previously published predictive scores. CONCLUSIONS: The HIAT2 score, which combines clinical and imaging variables, performed better than all previous scores in predicting poor outcome after IAT for anterior circulation large artery occlusions.

A Case of Subacute Stent Thrombosis.

A 67-year-old male presenting with an anterior ST-segment elevation myocardial infarction (STEMI) underwent stent placement in the left anterior descending coronary. The patient was discharged on an appropriate medical regimen containing dual antiplatelet therapy (DAPT). Four days later, the patient presented with repeat acute coronary syndrome symptomatology. Electrocardiogram demonstrated ongoing STEMI in the previously treated artery distribution. Emergency angiography revealed restenosis and total thrombotic occlusion. Post-intervention stenosis was 0% after aspiration thrombectomy and balloon angioplasty. Stent thrombosis is a high-mortality and therapeutically challenging condition requiring prepared clinicians who recognize predisposing risk factors and initiate early management.

Acute ischemic stroke treated with intravenous tissue plasminogen activator in a patient taking dabigatran with radiographic evidence of recanalization.

Dabigatran etexelate is a new oral direct thrombin inhibitor that has been approved by the US Food and Drug Administration to prevent stroke in patients with nonvalvular atrial fibrillation. A 51-year-old man with a history of atrial fibrillation who was taking dabigatran presented with an acute ischemic stroke. The patient had a normal international normalized ratio, activated partial thromboplastin time, and an elevated thrombin time of 26.4 seconds. Recanalization of the middle cerebral artery with intravenous tissue plasminogen activator was apparent on digital subtraction angiography, and there was no evidence of intracerebral hemorrhage on the repeat computed tomographic scan. This is the first report of a patient who was taking dabigatran etexilate and who had an ischemic stroke caused by a middle cerebral artery occlusion, with an elevated thrombin time and radiographic recanalization with intravenous tissue plasminogen activator without evidence of hemorrhagic transformation.

Pharmacological deep vein thrombosis prophylaxis does not lead to hematoma expansion in intracerebral hemorrhage with intraventricular extension.

BACKGROUND AND PURPOSE: Patients with intracerebral hemorrhage (ICH) are at high risk for development of deep venous thrombosis. Current guidelines state that low-dose subcutaneous low molecular weight heparin or unfractionated heparin may be considered at 3 to 4 days from onset. However, insufficient data exist on hematoma volume in patients with ICH before and after pharmacological deep venous thrombosis prophylaxis, leaving physicians with uncertainty regarding the safety of this practice. METHODS: We identified patients from our stroke registry (June 2003 to December 2007) who presented with ICH only or ICH+intraventricular hemorrhage and received either low molecular weight heparin subcutaneously or unfractionated heparin within 7 days of admission and had a repeat CT scan performed within 4 days of starting deep venous thrombosis prophylaxis. We calculated the change in hematoma volume from the admission and posttreatment CTs. Hematoma volume was calculated using the ABC/2 method and intraventricular hemorrhage volumes were calculated using a published method of hand drawn regions of interest. RESULTS: We identified 73 patients with a mean age of 63 years and median National Institutes of Health Stroke Scale score 11.5. The mean baseline total hematoma volume was 25.8 mL±23.2 mL. There was an absolute change in hematoma volume from pre- and posttreatment CT of -4.3 mL±11.0 mL. Two patients developed hematoma growth. Repeat analysis of patients given pharmacological deep venous thrombosis prophylaxis within 2 or 4 days after ICH found no increase in hematoma size. CONCLUSIONS: Pharmacological deep venous thrombosis prophylaxis given subcutaneously in patients with ICH and/or intraventricular hemorrhage in the subacute period is generally not associated with hematoma growth.

A report of fetal demise during therapeutic hypothermia after cardiac arrest.

BACKGROUND: Therapeutic hypothermia is becoming the standard-of-care for coma following out-of-hospital cardiac arrest. Pregnancy has been considered a contraindication for therapeutic hypothermia. METHODS: Case report. RESULTS: A 44-year-old woman presented after a witnessed out-of-hospital ventricular fibrillation cardiac arrest. She remained comatose upon hospital admission and was treated with induced hypothermia via surface cooling pads. An intrauterine pregnancy of 20 weeks gestation was discovered on admission. One day after admission, a stillborn fetus was spontaneously delivered. The patient made a good neurologic recovery and now lives at home with her family. CONCLUSION: During pregnancy, beneficence toward the pregnant woman must be the primary ethical guideline in emergent, life-threatening situations. Pregnancy should not be a contraindication to therapeutic hypothermia following cardiac arrest.

Intracranial hemorrhage following thrombolytic use for stroke caused by infective endocarditis.

BACKGROUND AND PURPOSE: Stroke is one of the most common neurological manifestations of infective endocarditis. The use of intravenous tissue plasminogen activator (t-PA) in the management of acute ischemic stroke is the accepted standard of practice. Current guidelines for intravenous (IV) t-PA therapy in acute ischemic stroke do not exclude patients with infective endocarditis. We present three patients who received IV t-PA for acute ischemic stroke in the setting of infective endocarditis and developed multifocal intracranial hemorrhage as a complication. CONCLUSION: Infective endocarditis related strokes are associated with a higher risk of hemorrhagic complications and our experience suggests that IV t-PA use may potentiate that risk.

Heart failure enigma in young man: the acute onset of a frequently encountered condition with an unexpected cause.

An 18-year-old male patient presented to the emergency department complaining of new onset chest pain, fever and orthopnoea. Initial workup was remarkable for elevated troponin, diffuse ST-segment elevation on ECG and chest X-ray with enlarged cardiac silhouette. Transthoracic echocardiogram (TTE) demonstrates severe biventricular concentric hypertrophy and pericardial effusion. Also, Coxsackie virus A and B titres were positive, concerning for a classic viral pericarditis. However, despite medical management, the patient became dyspnoeic and hypotensive. Impending cardiac tamponade was observed on repeat TTE, and pericardiocentesis was performed, complicated by pulseless electrical activity cardiac arrest, and ultimately patient requiring venoarterial extracorporeal membrane oxygenation support. Emergent endomyocardial biopsy showed no inflammatory process, and a skin biopsy of a small lesion in the right arm showed unexpected diagnosis of Epstein-Barr virus (+) natural killer/T-cell lymphoma. On initiation of chemotherapy, clinical improvement was observed as evidenced by improving ejection fraction, resolution of pericardial effusion and gradual decrease in myocardial hypertrophy.

Temperature Management in the Neurointensive Care Unit.

OPINION STATEMENT: Fever in the neurocritical care unit has a high prevalence and is associated with worse outcomes in patients with severe neurologic illness. While it is well accepted that fever is associated with worse outcomes in this patient population, it is unclear if aggressive temperature management will improve outcomes. Temperature should be monitored routinely in this high-risk population, fever worked up appropriately to identify infectious etiology, and reasonable measures taken to control elevated temperature. While infection is a common source of fever in patients with significant neurologic illness, the fever may also be exacerbated by the underlying brain injury. The clinician must decide at which point to initiate fever control measures, how aggressively to manage the fever, and which temperature to target for normothermia. Several pharmacological agents are available as first-line therapy. Depending on the degree and severity of the febrile response, advanced temperature-control devices should be added to pharmacological measures. Several types of temperature-control devices are available, including invasive (intravascular catheters) and noninvasive (external cooling pads) technologies. The clinician should utilize both pharmacologic and device-based temperature therapies to minimize the amount of time spent in a febrile state and help to mitigate the secondary brain injury brought on by fever.

CTP infarct core may predict poor outcome in stroke patients treated with IV t-PA.

BACKGROUND: Computerized tomography perfusion (CTP) has been widely studied in assessing physiological brain tissue parameters in patients with acute ischemic stroke (AIS). The utility of CTP to predict clinical outcome in patients with AIS treated with intravenous tissue plasminogen activator (IV t-PA) is controversial. We reviewed CTP data in AIS patients treated with IV t-PA to uncover potential predictors of clinical outcome. METHODS: We retrospectively identified AIS patients from our stroke registry (7/07 to 2/10) who underwent CTP on arrival and then received IV t-PA. A neuroradiologist blinded to outcome performed all CTP parameter measurements on a commercially available Siemens Neuro PCT workstation. Tissue at risk (TAR) was defined as the area of infarct territory with a relative time to peak (rTTP) greater than 4s. Non-viable tissue (NVT) was defined as the area of infarct territory with absolute cerebral blood volume (CBV) less than 2 ml/100g and cerebral blood flow (CBF) less than 12.7 ml/100g/min. Penumbra was defined as the area of (TAR) minus the area of (NVT). Excellent clinical outcome was defined as mRS (0-1), good clinical outcome was defined as mRS (0-2), and poor clinical outcome was defined as mRS (4-6), all measured at hospital discharge and 90 days if available. Recanalization data was obtained when available by comparing pre-thrombolytic CTA data and post-treatment MRA/CTA images by a single blinded radiologist. RESULTS: We identified 61 patients that met our inclusion criteria with a mean age of 68 (29-94), median NIHSS on admission of 13 (1-40), and median discharge mRS of 4 (0-6). Using multivariate logistic regression and ordinal logistic regression controlling for age and admission NIHSS, none of the CTP parameters were statistically associated with excellent or good clinical outcome (mRS<2). Using multivariate analysis controlling for age and admission NIHSS, NVT area>30 cm(2) (OR=5.12, CI: 0.95-27, p=0.05) was statistically associated with poor clinical outcome at discharge. NVT area ≥ 30 cm(2) was a potential predictor of poor outcome at discharge even when controlling for age and NIHSS. CONCLUSION: CTP parameters derived from commercially available software and published thresholds yield little predictive value for good clinical outcomes for AIS patients treated with IV t-PA but may be useful in predicting poor clinical outcome especially if the area of non-viable tissue is greater than 30 cm(2).

Design of a prospective, dose-escalation study evaluating the Safety of Pioglitazone for Hematoma Resolution in Intracerebral Hemorrhage (SHRINC).

RATIONALE : Preclinical work demonstrates that the transcription factor peroxisome proliferator-activated receptor gamma plays an important role in augmenting phagocytosis while modulating oxidative stress and inflammation. We propose that targeted stimulation of phagocytosis to promote efficient removal of the hematoma without harming surrounding brain cells may be a therapeutic option for intracerebral hemorrhage. AIMS : The primary objective is to assess the safety of the peroxisome proliferator-activated receptor gamma agonist, pioglitazone, in increasing doses for three-days followed by a maintenance dose, when administered to patients with spontaneous intracerebral hemorrhage within 24 h of symptom onset compared with standard care. We will determine the maximum tolerated dose of pioglitazone. STUDY DESIGN : This is a prospective, randomized, blinded, placebo-controlled, dose-escalation safety trial in which patients with spontaneous intracerebral hemorrhage are randomly allocated to placebo or treatment. The Continual Reassessment Method for dose finding is used to determine the maximum tolerated dose of pioglitazone. Hematoma and edema resolution is evaluated with serial magnetic resonance imaging (MRI) at specified time points. Functional outcome will be evaluated at three- and six-months. OUTCOMES : The primary safety outcome is mortality at discharge. Secondary safety outcomes include mortality at three-months and six-months, symptomatic cerebral edema, clinically significant congestive heart failure, edema, hypoglycemia, anemia, and hepatotoxicity. Radiographic outcomes will explore the time frame for resolution of 25%, 50%, and 75% of the hematoma. Clinical outcomes are measured by the National Institutes of Health Stroke Scale (NIHSS), the Barthel Index, modified Rankin Scale, Stroke Impact Scale-16, and EuroQol at three- and six-months.

Care of the stroke patient: routine management to lifesaving treatment options.

The management of the acute ischemic stroke patient spans the time course from the emergency evaluation and treatment period through to the eventual discharge planning phase of stroke care. In this article we evaluate the literature and describe what have become standard treatments in the care of the stroke patient. We will review the literature that supports the use of a dedicated stroke unit for routine stroke care which has demonstrated reduced rates of morbidity and mortality. Also reviewed is the use of glycemic control in the initial setting along with data supporting the use of prophylactic treatments options in order to aide in the prevention of life threatening medical complications. In addition, lifesaving treatments will be discussed in light of new literature demonstrating reduced mortality in large hemispheric stroke patients undergoing surgical decompressive surgery. Both medical and surgical treatment options are discussed and compared.