Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Ultraearly Intravenous Thrombolysis for Acute Ischemic Stroke in Mobile Stroke Unit and Hospital Settings.

Background and Purpose- Mobile stroke units (MSUs) are known to increase the proportion of acute ischemic stroke (AIS) patients treated with intravenous thrombolysis (IVT) in the first golden hour (GH) after onset compared with hospital settings (HS). However, because of the low number of AIS patients treated with intravenous thrombolysis within this ultraearly time window in conventional care, characteristics, and outcome of this subgroup of AIS patients have not been compared between MSU and HS. Methods- MSU-GH patients were selected from the Berlin-based MSU (STEMO [Stroke Emergency Mobile]), whereas HS-GH patients were selected from the SITS-EAST (Safe Implementation of Treatments in Stroke-East) registry. The outcome events of interest included the rates of favorable functional outcome (modified Rankin Scale scores of 0 or 1), distribution of the modified Rankin Scale scores, and mortality after 3 months between MSU-GH and HS-GH groups. Results- We identified 117 MSU-GH (38.4% of 305 MSU-treated patients) and 136 HS-GH (0.9% of 15 591 HS-treated patients) eligible patients without prestroke disability. No significant differences were documented in the rates of favorable functional outcome (51.3% versus 46.2%, P=0.487) and mortality (7.7% versus 9.9%, P=0.576) at 3 months, or in the distribution of 3-month modified Rankin Scale scores between the 2 groups ( P=0.196). In multivariable logistic regression analyses, adjusting for potential confounders, MSU treatment was not associated with a significantly different likelihood of favorable functional outcome (odds ratio, 1.84 for MSU patients; 95% CI, 0.86-3.96) or mortality (odds ratio, 0.95; 95% CI, 0.28-3.20) at 3 months. Conclusions- There is no evidence that safety and efficacy of ultraearly intravenous thrombolysis for AIS differs when used in MSUs or in HS.

Effects of Prehospital Thrombolysis in Stroke Patients With Prestroke Dependency.

BACKGROUND AND PURPOSE: Data on effects of intravenous thrombolysis on outcome of patients with ischemic stroke who are dependent on assistance in activities of daily living prestroke are scarce. Recent registry based analyses in activities of daily -independent patients suggest that earlier start of intravenous thrombolysis in the prehospital setting leads to better outcomes when compared with the treatment start in hospital. We evaluated whether these observations can be corroborated in patients with prestroke dependency. METHODS: This observational, retrospective analysis included all patients with acute ischemic stroke depending on assistance before stroke who received intravenous thrombolysis either on the Stroke Emergency Mobile (STEMO) or through conventional in-hospital care (CC) in a tertiary stroke center (Charité, Campus Benjamin Franklin, Berlin) during routine care. Prespecified outcomes were modified Rankin Scale scores of 0 to 3 and survival at 3 months, as well as symptomatic intracranial hemorrhage. Outcomes were adjusted in multivariable logistic regression. RESULTS: Between February 2011 and March 2015, 122 of 427 patients (28%) treated on STEMO and 142 of 505 patients (28%) treated via CC needed assistance before stroke. Median onset-to-treatment times were 97 (interquartile range, 69-159; STEMO) and 135 (interquartile range, 98-184; CC; P<0.001) minutes. After 3 months, modified Rankin Scale scores of 0 to 3 was observed in 48 STEMO patients (39%) versus 35 CC patients (25%; P=0.01) and 86 (70%, STEMO) versus 85 (60%, CC) patients were alive (P=0.07). After adjustment, STEMO care was favorable with respect to modified Rankin Scale scores of 0 to 3 (odds ratio, 1.99; 95% confidence interval, 1.02-3.87; P=0.042) with a nonsignificant result for survival (odds ratio, 1.73; 95% confidence interval, 0.95-3.16; P=0.07). Symptomatic intracranial hemorrhage occurred in 5 STEMO versus 12 CC patients (4.2% versus 8.5%; P=0.167). CONCLUSIONS: The results of this study suggest that earlier, prehospital (as compared with in-hospital) start of intravenous thrombolysis in acute ischemic stroke may translate into better clinical outcome in patients with prestroke dependency. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02358772.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization.

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

Vemurafenib and trametinib reduce expression of CTGF and IL-8 in V600EBRAF melanoma cells.

Clinical evidence has revealed that while RAS/RAF/MEK/ERK pathway is a crucial component of melanomagenesis, other signaling pathways can also contribute to the malignant growth and development of resistance to targeted therapies. We explored the response of V600EBRAF melanoma cells derived from surgical specimens and grown in stem cell medium to vemurafenib and trametinib, drugs targeting the activity of V600EBRAF and MEK1/2, respectively. Cell growth and apoptosis were monitored by real-time imaging system, immunophenotype and cell cycle by flow cytometry, gene expression by quantitative real-time PCR, immunoblotting and enzyme-linked immunosorbent assay. The V600EBRAF melanoma cell populations were diverse. Differences in morphology, pigmentation, cell cycle profiles, and immunophenotype were observed. At the molecular level, melanoma cells differed in the phosphorylation of ERK1/2, NF-κB, and ß-catenin, and expression of several relevant genes, including MITF-M, DKK1, CCND1, BRAF, CXCL8, and CTGF. Despite having different characteristics, melanoma cells responded similarly to vemurafenib and trametinib. Both drugs reduced ERK1/2 phosphorylation and percentages of cells expressing Ki-67 at high level, inhibited expression of CCND1 and induced cell cycle arrest in the Go/G1 phase. These expected cytostatic effects were accompanied by increased CD271 expression, a marker of stem-like cells. NF-κB activity was reduced by both drugs, however, not completely abolished, whereas the level of active ß-catenin was increased by drugs in three out of six cell populations. Interestingly, expression of IL-8 and CTGF was significantly reduced by treatment with vemurafenib and trametinib. Simultaneous inhibition of NF-κB activity and induction of ERK1/2 phosphorylation revealed that CTGF expression depends on ERK1/2 activity but not on NF-κB activity. Both, the positive effects of treatment with vemurafenib and trametinib such as the newly identified CTGF suppression and undesired effects such as increased CD271 expression suggesting selection of melanoma stem-like cells should be considered in the development of combination treatment for melanoma patients.

Glial Fibrillary Acidic Protein for Prehospital Diagnosis of Intracerebral Hemorrhage.

BACKGROUND: Both, acute ischemic stroke (AIS) and hemorrhage stroke (intracerebral hemorrhage, ICH) require early attention but different treatment strategies. Plasma glial fibrillary acidic protein (GFAP) levels were found to be elevated in ICH patients after they arrived in the hospital. Because treatment options differed, we sought to determine whether GFAP can be used to accurately differentiate between of AIS and ICH in the prehospital setting. METHODS: We assessed acute stroke patients in the Stroke Emergency Mobile (STEMO). STEMO is a stroke ambulance staffed by a specialized team including a neurologist and equipped with a computed tomography scanner plus a point-of-care laboratory. The STEMO ambulance is integrated in the emergency medical system of Berlin, Germany. Following prehospital stroke diagnosis, blood was drawn and subsequently analysed using research assays from Roche diagnostics. The clinical accuracy of plasma GFAP was tested using a cut-off value of 0.29 ng/ml. RESULTS: Blood samples of 74 patients were analysed. Twenty-five patients had ICH (mean age 69 ± 11 years, median National Institutes of Health Stroke Scale (NIHSS) 15) and 49 IS (mean age 75 ± 10 years, median NIHSS 6). Nine ICH (0 IS patients) had GFAP-levels above 0.29 ng/ml. The sensitivity and specificity of GFAP for differentiating between ICH and AIS were 36.0 and 100%. The sensitivity for ICH volume >15 ml was 61.5%. ICH patients without GFAP elevation had significantly smaller hemorrhage volumes (median 4.5 vs. 37.6 ml, p = 0.004) and were less likely to deteriorate (19 vs. 56%, p = 0.087). CONCLUSIONS: GFAP levels >0.29 ng/ml were seen only in ICH, thus confirming the diagnosis of ICH during prehospital care. However, sensitivity is low particularly in smaller hemorrhages.

Influence of Distance to Scene on Time to Thrombolysis in a Specialized Stroke Ambulance.

BACKGROUND AND PURPOSE: Specialized computed tomography-equipped stroke ambulances shorten time to intravenous thrombolysis in acute ischemic stroke by starting treatment before hospital arrival. Because of longer travel-time-to-scene, time benefits of this concept are expected to diminish with longer distances from base station to scene. METHODS: We used data from the Prehospital Acute Neurological Treatment and Optimization of Medical Cares in Stroke (PHANTOM-S) trial comparing time intervals between patients for whom a specialized stroke ambulance (stroke emergency mobile) was deployed and patients with conventional emergency medical service. Expected times from base station to scene had been calculated beforehand using computer algorithms informed by emergency medical service routine data. Four different deployment zones with-75% probability-expected arrival within 4, 8, 12, and 16 minutes and total population coverage of ≈1.3 million inhabitants were categorized for stroke emergency mobile deployment. We analyzed times from alarm-to-arrival at scene, to start of intravenous thrombolysis and from onset-to-intravenous thrombolysis. RESULTS: Corresponding to the size of the respective catchment zone, the number of patients cared increased with distance (zone 1: n=30, zone 2: n=127, zone 3: n=156, and zone 4: n=217). Although time to stroke emergency mobile arrival increased with distance (mean: 8.0, 12.5, 15.4, and 18.4 minutes in zones 1-4), time from alarm-to-intravenous thrombolysis (mean: 41.8 versus 76.5; 50.2 versus 79.1; 54.5 versus 76.6; and 59.3 versus 78.0 minutes, respectively; all P<0.01) remained shorter in the stroke emergency mobile group across all zones. CONCLUSIONS: In a metropolitan area such as Berlin, time benefits justify a specialized stroke ambulance service up to a mean travel time of 18 minutes from base station. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01382862.

[Not Available]. / Mieszanki ziolowe o dzialaniu przeciwgoraczkowym jako przyklady tradycyjnego leku roslinnego stosowanego w populacji pediatrycznej.

Herbal medicines with antipyretic action have a long tradition of use in Polish phytotherapy. Over the years, they have proven both their efficacy and safety in a feverishness, which very often have accompanied the upper respiratory tract infections in children. Leading active ingredient of these products is the willow bark (Cortex salicis), which has been the active ingredient in both, the herbal mixture Pyrosan placed on the market in the 50's of last century and Pyrosal syrup marketed in the year 1989. Other herbal substances used in such preparations are elder flower, lime flower and meadowsweet flower. The usage of a pharmaceutical form of syrup is prerequisite when the main group of the patients are children. Such products are continuously in use for nearly 30 years in Poland.

Copeptin Levels in Patients With Acute Ischemic Stroke and Stroke Mimics.

BACKGROUND AND PURPOSE: Copeptin levels are increased in patients diagnosed with stroke and other vascular diseases. Copeptin elevation is associated with adverse outcome, predicts re-events in patients with transient ischemic attack and is used in ruling-out acute myocardial infarction. We evaluated whether copeptin can also be used as a diagnostic marker in the prehospital stroke setting. METHODS: We prospectively examined patients with suspected stroke on the Stroke Emergency Mobile-an ambulance that is equipped with computed tomography and point-of-care laboratory. A blood sample was taken from patients immediately after arrival. We analyzed copeptin levels in patients with final hospital-based diagnosis of stroke or stroke mimics as well as in vascular or nonvascular patients. In addition, we examined the associations of symptom onset with copeptin levels and the prognostic value of copeptin in patients with stroke. RESULTS: Blood samples of 561 patients were analyzed. No significant differences were seen neither between cerebrovascular (n=383) and other neurological (stroke mimic; n=90) patients (P=0.15) nor between vascular (n=391) and nonvascular patients (n=170; P=0.57). We could not detect a relationship between copeptin levels and time from onset to blood draw. Three-month survival status was available in 159 patients with ischemic stroke. Copeptin levels in nonsurviving patients (n=8: median [interquartile range], 27.4 [20.2-54.7] pmol/L) were significantly higher than in surviving patients (n=151: median [interquartile range], 11.7 [5.2-30.9] pmol/L; P=0.024). CONCLUSIONS: In the prehospital setting, copeptin is neither appropriate to discriminate between stroke and stroke mimic patients nor between vascular and nonvascular patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01382862. The Pre-Hospital Acute Neurological Therapy and Optimization of Medical Care in Stroke Patients study (PHANTOM-S) was registered (NCT01382862). This sub-study was observational and not registered separately, therefore.

Improved prehospital triage of patients with stroke in a specialized stroke ambulance: results of the pre-hospital acute neurological therapy and optimization of medical care in stroke study.

BACKGROUND AND PURPOSE: Specialized management of patients with stroke is not available in all hospitals. We evaluated whether prehospital management in the Stroke Emergency Mobile (STEMO) improves the triage of patients with stroke. METHODS: STEMO is an ambulance staffed with a specialized stroke team and equipped with a computed tomographic scanner and point-of-care laboratory. We compared the prehospital triage of patients with suspected stroke at dispatcher level who either received STEMO care or conventional care. We assessed transport destination in patients with different diagnoses. Status at hospital discharge was used as short-term outcome. RESULTS: From May 2011 to January 2013, 1804 of 6182 (29%) patients received STEMO care and 4378 of 6182 (71%) patients conventional care. Two hundred forty-five of 2110 (11.6%) patients with cerebrovascular events were sent to hospitals without Stroke Unit in conventional care when compared with 48 of 866 (5.5%; P<0.01%) patients in STEMO care. In patients with ischemic stroke, STEMO care reduced transport to hospitals without Stroke Unit from 10.1% (151 of 1497) to 3.9% (24 of 610; P<0.01). The delivery rate of patients with intracranial hemorrhage to hospitals without neurosurgery department was 43.0% (65 of 151) in conventional care and 11.3% (7 of 62) in STEMO care (P<0.01). There was a slight trend toward higher rates of patients discharged home in neurological patients when cared by STEMO (63.5% versus 60.8%; P=0.096). CONCLUSIONS: The triage of patients with cerebrovascular events to specialized hospitals can be improved by STEMO ambulances. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01382862.

Hyperintense acute reperfusion marker is associated with higher contrast agent dosage in acute ischaemic stroke.

OBJECTIVE: The hyperintense acute reperfusion marker (HARM) on fluid-attenuated inversion recovery (FLAIR) images is associated with blood-brain barrier (BBB) permeability changes. The aim of this study was to examine the influence of contrast agent dosage on HARM incidence in acute ischaemic stroke patients. METHODS: We prospectively included 529 acute ischaemic stroke patients (204 females, median age 71 years). Patients underwent a first stroke-MRI within 24 hours from symptom onset and had a follow-up on day 2. The contrast agent Gadobutrol was administered to the patients for perfusion imaging or MR angiography. The total dosage was calculated as ml/kg body weight and ranged between 0.04 and 0.31 mmol/kg on the first examination. The incidence of HARM was evaluated on day 2 FLAIR images. RESULTS: HARM was detected in 97 patients (18.3%). HARM incidence increased significantly with increasing dosages of Gadobutrol. Also, HARM positive patients were significantly older. HARM was not an independent predictor of worse clinical outcome, and we did not find an association with increase risk of haemorrhagic transformation. CONCLUSIONS: A higher dosage of Gadobutrol in acute stroke patients on initial MRI is associated with increased HARM incidence on follow-up. MRI studies on BBB should therefore standardize contrast agent dosages. KEY POINTS: • Hyperintense acute reperfusion marker on MRI indicates blood-brain barrier disruption. • This observational study on stroke patients characterizes HARM. • Incidence depends on contrast agent dosage on the previous day. • HARM is also associated with older age and poor kidney function. • Interpretation of HARM must take dosage into consideration.

Elevated levels of hemoglobin A1c are associated with cerebral white matter disease in patients with stroke.

BACKGROUND AND PURPOSE: This study was conducted to investigate the association of cerebral white matter disease (WMD) on MRI with vascular risk factors and laboratory findings in consecutive first acute ischemic stroke patients. METHODS: Acute ischemic stroke patients underwent MRI ≤24 hours after stroke onset and follow-up on day 2. WMD was scored on fluid attenuated inversion recovery MRI according to the Wahlund score. Vascular risk factors and laboratory parameters were assessed during hospital stay. Univariate and multiple logistic regression analyses were performed. RESULTS: We included 512 patients with first acute ischemic stroke (mean age, 68.5 [SD, 13.2] years; 192 women (37.5%); median National Institutes of Health Stroke Scale on admission, 3 [interquartile range, 1-6]; and median Wahlund score, 4 [interquartile range, 2-9]). WMD was present in 460 (89.8%) patients. In univariate analysis, age, arterial hypertension, reduced estimated glomerular filtration rate, hemoglobin A1c (HbA1c) levels, diabetes mellitus, and female sex were associated with the presence of WMD (P<0.05). In multiple regression analysis, age, arterial hypertension, and elevated levels of HbA1c (P<0.05) remained independently associated with the extent of WMD. CONCLUSIONS: Among known risk factors, higher levels of HbA1c were associated with cerebral WMD in stroke patients. This may suggest that chronic disturbance of glycemia measured by HbA1c plays a role in the pathophysiology of WMD. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00715533.

Number of cerebral microbleeds and risk of intracerebral hemorrhage after intravenous thrombolysis.

BACKGROUND AND PURPOSE: Cerebral microbleeds (CMBs) are found in a substantial proportion of patients with ischemic stroke eligible for treatment with intravenous thrombolysis. Until now, there is limited data on the impact of multiple CMBs on occurrence of intracerebral hemorrhage (ICH) after intravenous thrombolysis. METHODS: Between 2008 and 2013, all patients receiving MRI-based intravenous thrombolysis were identified within our prospective thrombolysis register. Number of CMBs was rated on pretreatment T2*-weighted MRI by a rater blinded to clinical data and follow-up. Outcomes of interest were occurrence of symptomatic ICH (sICH) and parenchymal hemorrhage (PH). RESULTS: Among 326 included patients, 52 patients had a single CMB (16.0%), 19 had 2 to 4 CMBs (5.8%), and 10 had ≥5 CMBs (3.1%). Frequency of sICH/PH was 1.2%/5.7% in patients without CMBs, 3.8%/3.8% in patients with a single CMB, 10.5%/21.1% in patients with 2 to 4 CMBs, and 30.0%/30.0% in patients with ≥5 CMBs, respectively (each P for trend<0.01). The unadjusted odds ratio per additional CMB for sICH was 1.19 (95% confidence interval, 1.07-1.33; P<0.01) and for PH was 1.13 (95% confidence interval, 1.03-1.24; P=0.01). Compared with patients without CMBs, both patients with 2 to 4 CMBs (P=0.02/P=0.02) and patients with ≥5 CMBs (P<0.01/P<0.01) had significantly increased odds ratios for sICH and PH, whereas in patients with a single CMB, odds ratios were not significantly increased (P=0.21/P=0.59). The association of CMB burden with sICH/PH remained significant after adjustment for possible confounders (age, age-related white matter changes score, atrial fibrillation, onset-to-treatment time, prior statin use, and systolic blood pressure on admission). CONCLUSIONS: Our findings indicate a higher risk of sICH and PH after intravenous thrombolysis when multiple CMBs are present, with a graded relationship to increasing baseline CMB number.

Early time course of FLAIR signal intensity differs between acute ischemic stroke patients with and without hyperintense acute reperfusion marker.

BACKGROUND: In animal models of stroke, the time course of blood-brain barrier (BBB) disruptions has been elaborately studied. In human patients, leakage of gadolinium into cerebrospinal fluid (CSF) space, visualized on MRI fluid attenuated inversion recovery (FLAIR) images, is considered a sign of BBB disruptions. It was termed 'hyperintense acute reperfusion marker' (HARM) and was associated with hemorrhages. However, the time course of the leakage is unknown and difficult to study in human patients. Also, the association of HARM with signal intensities and enhancement in the parenchyma on FLAIR images has not been thoroughly researched. METHODS: We analyzed imaging data of acute ischemic stroke patients who underwent repetitive MRI examinations within the first 36 h after the time of symptom onset. HARM was evaluated on FLAIR images. Regions of interest (ROI) of the hyperintensities on diffusion-weighted imaging (DWI) were determined for each time point and mirrored to the contralateral side. The ROI were furthermore corrected for CSF-filled space, using apparent diffusion coefficient (ADC) images. The corrected ROI were used to determine mean signal intensities of the lesions relative to the contralateral side on FLAIR, ADC and B0 images for each time point. RESULTS: The 18 included patients (5 females; median age: 69 years; median NIHSS score: 5) received 3-5 MRI examinations on the first day and 1-2 examinations on day 2 after stroke. Eight of the patients (44.4%) showed HARM on at least 1 examination. In 6 of these patients, HARM was already seen at the second examination, at the earliest 3.5 h after symptom onset. The HARM-positive patients had higher relative signal intensities (rSI) on FLAIR images in the parenchyma corresponding to the DWI-positive tissue compared with the HARM-negative patients. This difference between groups was statistically significant for the 2nd and 3rd examination (medians of 4.31 and 6.37 h from symptom onset, p < 0.001 and p = 0.005, respectively). No significant difference in rSI between groups was seen for ADC or B0 images. CONCLUSION: HARM does not only represent a contrast medium leakage from the pial system into the CSF space. It is accompanied by a markedly increased rSI in the early ischemic lesion on FLAIR images, which is likely due to parenchymal enhancement. The lack of differences on B0 images excludes a pure T2 effect.

Effect of the use of ambulance-based thrombolysis on time to thrombolysis in acute ischemic stroke: a randomized clinical trial.

IMPORTANCE: Time to thrombolysis is crucial for outcome in acute ischemic stroke. OBJECTIVE: To determine if starting thrombolysis in a specialized ambulance reduces delays. DESIGN, SETTING, AND PARTICIPANTS: In the Prehospital Acute Neurological Treatment and Optimization of Medical care in Stroke Study (PHANTOM-S), conducted in Berlin, Germany, we randomly assigned weeks with and without availability of the Stroke Emergency Mobile (STEMO) from May 1, 2011, to January 31, 2013. Berlin has an established stroke care infrastructure with 14 stroke units. We included 6182 adult patients (STEMO weeks: 44.3% male, mean [SD] age, 73.9 [15.0] y; control weeks: 45.2% male, mean [SD] age, 74.3 [14.9] y) for whom a stroke dispatch was activated. INTERVENTIONS: The intervention comprised an ambulance (STEMO) equipped with a CT scanner, point-of-care laboratory, and telemedicine connection; a stroke identification algorithm at dispatcher level; and a prehospital stroke team. Thrombolysis was started before transport to hospital if ischemic stroke was confirmed and contraindications excluded. MAIN OUTCOMES AND MEASURES: Primary outcome was alarm-to-thrombolysis time. Secondary outcomes included thrombolysis rate, secondary intracerebral hemorrhage after thrombolysis, and 7-day mortality. RESULTS: Time reduction was assessed in all patients with a stroke dispatch from the entire catchment area in STEMO weeks (3213 patients) vs control weeks (2969 patients) and in patients in whom STEMO was available and deployed (1804 patients) vs control weeks (2969 patients). Compared with thrombolysis during control weeks, there was a reduction of 15 minutes (95% CI, 11-19) in alarm-to-treatment times in the catchment area during STEMO weeks (76.3 min; 95% CI, 73.2-79.3 vs 61.4 min; 95% CI, 58.7-64.0; P < .001). Among patients for whom STEMO was deployed, mean alarm-to-treatment time (51.8 min; 95% CI, 49.0-54.6) was shorter by 25 minutes (95% CI, 20-29; P < .001) than during control weeks. Thrombolysis rates in ischemic stroke were 29% (310/1070) during STEMO weeks and 33% (200/614) after STEMO deployment vs 21% (220/1041) during control weeks (differences, 8%; 95% CI, 4%-12%; P < .001, and 12%, 95% CI, 7%-16%; P < .001, respectively). STEMO deployment incurred no increased risk for intracerebral hemorrhage (STEMO deployment: 7/200; conventional care: 22/323; adjusted odds ratio [OR], 0.42, 95% CI, 0.18-1.03; P = .06) or 7-day mortality (9/199 vs 15/323; adjusted OR, 0.76; 95% CI, 0.31-1.82; P = .53). CONCLUSIONS AND RELEVANCE: Compared with usual care, the use of ambulance-based thrombolysis resulted in decreased time to treatment without an increase in adverse events. Further studies are needed to assess the effects on clinical outcomes. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01382862.

Development and validation of a dispatcher identification algorithm for stroke emergencies.

BACKGROUND AND PURPOSE: Recent innovations such as CT installation in ambulances may lead to earlier start of stroke-specific treatments. However, such technically complex mobile facilities require effective methods of correctly identifying patients before deployment. We aimed to develop and validate a new dispatcher identification algorithm for stroke emergencies. METHODS: Dispatcher identification algorithm for stroke emergencies was informed by systematic qualitative analysis of the content of emergency calls to ambulance dispatchers for patients with stroke or transient ischemic attack (N=117) and other neurological (N=39) and nonneurological (N=51) diseases (Part A). After training of dispatchers, sensitivity and predictive values were determined prospectively in patients admitted to Charité hospitals by using the discharge diagnosis as reference standard (Part B). RESULTS: Part A: Dysphasic/dysarthric symptoms (33%), unilateral symptoms (22%) and explicitly stated suspicion of stroke (47%) were typically identified in patients with stroke but infrequently in nonstroke cases (all <10%). Convulsive symptoms (41%) were frequent in other neurological diseases but not strokes (3%). Pain (26%) and breathlessness (31%) were often expressed in nonneurological emergencies (6% and 7% in strokes). Part B: Between October 15 and December 16, 2010, 5774 patients were admitted by ambulance with 246 coded with final stroke diagnoses. Sensitivity of dispatcher identification algorithm for stroke emergencies for detecting stroke was 53.3% and positive predictive value was 47.8% for stroke and 59.1% for stroke and transient ischemic attack. Of all 275 patients with stroke dispatcher codes, 215 (78.5%) were confirmed with neurological diagnosis. CONCLUSIONS: Using dispatcher identification algorithm for stroke emergencies, more than half of all patients with stroke admitted by ambulance were correctly identified by dispatchers. Most false-positive stroke codes had other neurological diagnoses.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay.

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.

Probability assessment of intracerebral hemorrhage in prehospital emergency patients.

BACKGROUND: Routing of patients with intracerebral hemorrhage (ICH) and acute ischemic stroke (AIS) to the most appropriate hospital is challenging for emergency medical services particularly when specific treatment options are only provided by specialized hospitals and determination of the exact diagnosis is difficult. We aimed to develop a prehospital score - called prehospital-intracerebral hemorrhage score (ph-ICH score) - to assist in discriminating between both conditions. METHODS: The ph-ICH score was developed with data from patients treated aboard a mobile stroke unit in Berlin, Germany, between 2011 and 2013 (derivation cohort) and in 2018 (validation cohort). Diagnosis of ICH or AIS was established using clinical data and neuroradiological cerebral imaging. Diagnostic accuracy was measured with significance testing, Cohen's d and receiver-operating-characteristics. RESULTS: We analyzed 416 patients (32 ICH, 224 AIS, 41 transient ischemic attack, 119 stroke mimic) in the derivation cohort and 285 patients (33 ICH and 252 AIS) in the validation cohort. Systolic blood pressure, level of consciousness and severity of neurological deficits (i. e. certain items of the National Institutes of Health Stroke Scale) were used to calculate the ph-ICH score that showed higher values in the ICH compared to the AIS group (derivation cohort: 1.8 ± 1.2 vs. 1.0 ± 0.9 points; validation cohort: 1.8 ± 0.9 vs. 0.8 ± 0.7 points; d = 0.9 and 1.4, both p < 0.01). Receiver-operating-characteristics showed fair and good accuracy with an area under the curve of 0.71 for the derivation and 0.81 for the validation cohort. CONCLUSIONS: The ph-ICH score can assist medical personnel in the field to assess the likelihood of ICH and AIS in emergency patients.