Pre-hospital stroke screening and notification of patients with reperfusion-eligible acute ischaemic stroke using modified Face Arm Speech Time test.

OBJECTIVES: To investigate the effects of pre-hospital stroke screening and notification on reperfusion therapy for patients with acute ischaemic stroke. METHODS: Pre-hospital stroke screening criteria were established based on a modified version of the Face Arm Speech Time (FAST) test. Screening was performed during ambulance transport by emergency medical service (EMS) personnel who completed a 2-hour training session on stroke screening. Temporal trends affecting acute ischaemic stroke investigation and intervention were compared before and after implementation of the pre-hospital screening. RESULTS: From July 2018 to October 2019, 298 patients with suspected stroke were screened by EMS personnel during ambulance transport prior to hospital arrival. Of these 298 patients, 213 fulfilled the screening criteria, 166 were diagnosed with acute stroke, and 32 received reperfusion therapy. The onset-to-door time was shortened by more than 1.5 hours (100.6 min vs 197.6 min, P<0.001). The door-to-computed tomography time (25.6 min vs 32.0 min, P=0.021), door-to-needle time (49.2 min vs 70.1 min, P=0.003), and door-to-groin puncture time for intra-arterial mechanical thrombectomy (126.7 min vs 168.6 min, P=0.04) were significantly shortened after implementation of the pre-hospital screening and notification, compared with historical control data of patients admitted from January 2018 to June 2018, before implementation of the screening system. CONCLUSION: Implementation of pre-hospital stroke screening using criteria based on a modified version of the FAST test, together with pre-arrival notification, significantly shortened the door-to-reperfusion therapy time for patients with ischaemic stroke. Pre-hospital stroke screening during ambulance transport by EMS personnel who complete a 2-hour focused training session is effective for identifying reperfusion-eligible patients with stroke.

Prehospital electrocardiogram shortens ischaemic time in patients with ST-segment elevation myocardial infarction.

INTRODUCTION: Total ischaemic time should be shortened as much as possible in patients with ST-segment elevation myocardial infarction (STEMI). This study evaluated whether prehospital 12-lead electrocardiogram (ECG) could shorten system delay in STEMI management. METHODS: From November 2015 to November 2017, 15 ambulances equipped with X Series Monitor/ Defibrillator (Zoll Medical Corporation) were used in the catchment area of Queen Mary Hospital, Hong Kong. Prehospital ECG was performed for patients with chest pain; the data were tele-transmitted to attending emergency physicians at the Accident and Emergency Department (AED) for rapid assessment. Data from patients with STEMI who were transported by these 15 ambulances were compared with data from patients with STEMI who were transported by ambulances without prehospital ECG or who used self-arranged transport. RESULTS: Data were analysed from 197 patients with STEMI. The median patient delay for activation of the emergency response system was 90 minutes; 12% of patients experienced a delay of >12 hours. There was a significant difference in delay between patients transported by ambulance and those who used self-arranged transport (P<0.001). For system delay, the use of prehospital ECG shortened the median time from ambulance on scene to first ECG (P<0.001). When performed upon ambulance on scene, prehospital ECG was available 5 minutes earlier than if performed in ambulance compartment before departure. Use of prehospital ECG significantly shortened AED door-to-triage time, AED door-to-first AED ECG time, AED door-to-physician consultation time, and length of stay in the AED (P<0.001 for all comparisons). CONCLUSION: Prehospital ECG shortened ischaemic time prior to hospital admission.

Prehospital 12-lead electrocardiogram for patients with chest pain: a pilot study.

INTRODUCTION: After ST-segment elevation myocardial infarction (STEMI), it is vital to shorten reperfusion time. This study examined data from a pilot project to shorten the door-to-balloon (D2B) time by using prehospital 12-lead electrocardiogram (ECG). METHODS: Fifteen ambulances equipped with X Series® Monitor/Defibrillator (Zoll Medical Corporation) were deployed to the catchment area of Queen Mary Hospital, Hong Kong, from November 2015 to December 2016. For patients with chest pain, prehospital 12-lead ECG was performed and tele-transmitted to attending physicians at the accident and emergency department for immediate interpretation. The on-call cardiologist was called before patient arrival if STEMI was suspected. Data from this group of patients with STEMI were compared with data from patients with STEMI who were transported by ambulances without prehospital ECG or by self-arranged transport. RESULTS: From 841 patients with chest pain, 731 gave verbal consent and prehospital ECG was performed and transmitted. Of these, 25 patients with clinically diagnosed STEMI required emergency coronary angiogram with or without primary percutaneous coronary intervention. The mean D2B time for these 25 patients (93 minutes) was significantly shorter (P=0.003) than that for 58 patients with STEMI transported by ambulances without prehospital ECG (112 minutes) and that for 41 patients with STEMI with self-arranged transport (138 minutes). However, shorter reperfusion time was only recorded during daytime hours (08:00-17:59). No statistically significant difference in 30-day mortality was found. CONCLUSION: Prehospital ECG is technologically feasible in Hong Kong and shortens the D2B time. However, shorter reperfusion time was only recorded during daytime hours.

A self-augmenting gene expression cassette for enhanced and sustained transgene expression in the presence of proinflammatory cytokines.

Viral promoters can yield high gene expression levels yet tend to be attenuated in vivo by host proinflammatory cytokines. Prolonged transgene expression can be obtained using constitutive cellular promoters. However, levels of transgene expression driven by cellular promoters are insufficient for effective therapy. We designed a novel self-augmenting gene expression cassette in which the transgene product can induce an endogenous transcription factor to enhance the activity of a weak cellular promoter driving its expression. Using the cellular major histocompatibility complex class I (H-2K(b)) promoter to drive the interferon (IFN-gamma) cytokine gene, we show that the H-2K(b) promoter, although exhibiting much lower basal activity, yields higher IFN-gamma production than the CMV promoter 2 days after transfection. IFN-gamma expression driven by the H-2K(b) promoter also lasts longer than that driven by the cytomegalovirus promoter. Our data demonstrate that the self-augmenting strategy provides a promising approach to achieve high and sustained transgene expression in vivo.

Novel amplifier expression vectors producing higher levels of IL-2 led to slower tumor growth and longer survival in vivo.

Sufficient levels of gene expression are required for effective gene therapy. One of the major obstacles in gene therapy is the low transgene expression obtained from currently available vector systems. To address this issue, we employed a transcriptional amplifier strategy in a single construct to enhance transgene expression. In the amplifier vectors (pHi-1 and pHi-2), the strong CMV promoter was used to drive a transcriptional factor, Tat, which could transactivate a second promoter (HIV1 LTR or HIV2 LTR) located in the same construct driving the gene of interest. Using the human interleukin-2 (IL-2) cytokine gene, our data showed that the pHi-1/2 amplifier vectors could produce significantly higher IL-2 levels in human lung cancer cells (A549) and breast cancer cells (MCF-7) than that obtained by directly using the CMV promoter alone. Injection of pHi-2-IL-2-modified Lewis Lung (LL/2) tumor clones led to significantly slower tumor growth and longer survival in mice compared to those injected with either CMV promoter driven IL-2 clones or the parental tumor cells. Our results demonstrated that the transcriptional amplifier-based expression cassettes could be very useful in applications where high levels of gene expression are difficult to achieve.

Heat-inducible vectors for use in gene therapy.

The objectives of this study were to quantity and compare the activities of a minimal heat shock (HS) promoter and other promoters used in gene therapy applications, and to identify strategies to amplify the heat inducibility of therapeutic genes. Human tumour cells were transiently or stably transfected with the HS promoter driving expression of reporter genes. HS promoter activity was induced transiently, with maximum activity 16-24 h after HS, and was dependent on temperature. The activity of the minimal HS promoter was similar, after 42 degrees C HS for 1 h, to that of the cytomegalovirus (CMV) promoter. To determine if the HS promoter could be used to activate a second conditional promoter, cells were transiently transfected with vectors containing both the HS and human immunodeficiency virus type 1 (HIV1) promoters. When the IL-2 gene was placed downstream of the HIV1 promoter. IL-2 production was temperature-independent. The addition of the HIV tat gene downstream of the HS promoter caused IL-2 to be induced more than 3 fold after a single 42 degrees C HS. These data indicate that the minimal HS promoter, following activation by clinically attainable temperatures (< or = 42 degrees C), can drive expression of therapeutic genes at levels comparable to the CMV promoter and be used in conjunction with a second conditional promoter to drive temperature-dependent, gene expression.

Construction of new amplifier expression vectors for high levels of IL-2 gene expression.

The success of IL-2 gene therapy in cancer is in part dependent on the development of high level IL-2 gene expression vectors. Currently, expression vectors based on the human cytomegalovirus (CMV) promoter give the highest levels of expression. We have attempted to construct new IL-2 expression vectors to test whether gene expression can be further increased. The first approach was to use the new SR-alpha promoter to control IL-2 gene expression. The second approach was to combine the Tat transcription activator gene and the HIV 1 and 2 promoters in the same construct so that the levels of gene expression can be amplified. Transient transfection results using the human colon cancer cell line SW480 showed that the SR-alpha promoter yields similar levels of activity as the CMV promoter. However, the HIV 1 and 2 promoter-based amplifier constructs produced 11 and 28 times more secreted IL-2 than the CMV promoter control. The augmented activity of the amplifier constructs was dependent on the presence of the Tat gene and the transcriptional units must be placed in the same orientation. Reducing the size of the vectors by elimination of the neomycin selectable marker did not increase the activity of the constructs.

Differential expression of matrix metalloproteinases in activated c-ras-Ha-transfected immortalized human keratinocytes.

Elevated expression of matrix metalloproteinases (MMPs), a family of secreted proteinases that degrade matrix components of basement membranes and connective tissues, is strongly correlated with malignant expression in various human epithelial cancers and epithelial cancer cell lines. We have tested whether elevated levels of MMP expression are also associated with malignant progression in human cutaneous squamous cell carcinoma. Constitutive levels of expression of steady-state mRNA and of secreted protein encoded by three MMP genes (matrilysin, gelatinases A and B) were compared in a unique in vitro model of human skin carcinogenesis. This model is composed of the parental immortalized non-tumorigenic human keratinocyte line (HaCaT), and three activated c-Harvey-ras-oncogene transfected variants (A-4, I-7 and II-4). Although clone A-4 is non-tumorigenic, clones I-7 and II-4 exhibit benign and malignant tumorigenic phenotypes, respectively, after subcutaneous injection into athymic nude mice. Northern blot, Western blot, and zymogram analyses revealed three MMP-specific patterns of expression. Constitutive matrilysin mRNA expression was markedly increased in the I-7 cells compared with HaCaT, A-4 or II-4 cells. Secreted promatrilysin was distinctly increased in the tumorigenic I-7 and II-4 cells compared with the non-tumorigenic HaCaT and A-4 cells. Gelatinase A mRNA and secreted gelatinase A protein levels were increased in each transfectant compared with HaCaT. Both active and inactive forms of gelatinase A were detected. Gelatinase B transcripts were not detected, but an EDTA-inhibitable gelatinase activity comigrating with gelatinase B was moderately enhanced in both tumorigenic variants compared with the non-tumorigenic cells. Because promatrilysin and 92-kDa gelatinase secretion were increased in both benign and malignant tumorigenic cells, and not related to invasiveness in this model, it is concluded that enhanced constitutive expression of these two MMPs is associated with acquisition of the tumorigenic phenotype, before acquisition of the malignant phenotype.

The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.

Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane.

v-jun oncogene suppresses both phorbol ester-induced cell invasion and stromelysin gene expression in a mouse papilloma cell line.

The viral jun (v-jun) oncogene encodes a transcription factor that can participate in the transactivation of genes through the AP-1 complex. Evidence indicates that the ability of v-jun to transform cells and stimulate transcription depends on the cell type. We have asked whether expression of the v-jun gene in benign tumor forming mouse keratinocytes that already express an activated c-rasHa oncogene would cause malignant progression. Our results showed that the v-jun transfection did not result in malignant progression; instead, we made the unexpected observation that the ability of these cells to invade reconstituted basement membrane matrix (in vitro) in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was suppressed. This phenomenon could, in part, be explained by the suppression of the induction by phorbol ester of expression of the metalloproteinase, stromelysin (transin). Of interest was the finding that 12-O-tetradecanoylphorbol-13-acetate induction of other cellular genes known to be regulated by AP-1 was not inhibited in the benign tumor cells expressing v-jun.

New model for 70 kDa heat-shock proteins' potential mechanisms of function.

The 70 kDa family of heat-shock proteins (hsp 70s and hsc 70s) can facilitate protein transport to several organelles. This process is thought to involve electrostatic interactions between hsp 70s and the cellular protein targeting sequences. Analysis of the highly conserved structural and functional properties of the hsp 70 family indicated that they may cross-link cellular proteins to the actin microfilament network. Direct experimental support for this hypothesis was provided by the finding that hsp 70 is constitutively bound to actin through hydrophobic interactions. The cross-linker model may provide an explanation for the mechanism by which the cytoskeletal matrix could mediate various cellular processes.

Characterization of cDNA and genomic sequences encoding rabbit ELAM-1: conservation of structure and functional interactions with leukocytes.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is a cytokine-inducible endothelial cell surface glycoprotein involved in the adherence of neutrophils. ELAM-1 belongs to the selectin family of cell-surface molecules characterized by the general structure of an amino-terminal lectin domain followed by an epidermal growth factor domain, a variable number of complement regulatory elements, a single transmembrane sequence, and a short cytoplasmic tail. To study the in vivo regulation and expression of ELAM-1, we have isolated a complementary DNA (cDNA) clone encoding the rabbit homolog of human ELAM-1. The nucleotide sequence of the rabbit cDNA as well as its deduced amino acid sequence display extensive conservation compared to the human sequences. Rabbit ELAM-1 contains the characteristic protein domain organization of the selectin gene family and shares 74% amino acid identity with its human counterpart. However, rabbit ELAM-1 contains five complement regulatory elements whereas the human protein has six of these elements. Characterization of the genomic sequence encoding rabbit ELAM-1 indicated that individual extracellular protein domains are encoded by distinct exons. The genomic organization of rabbit ELAM-1 parallels that found for the human ELAM-1 gene and is similar to the pattern observed for other selectin family members (GMP-140, Lam-1), consistent with the hypothesis that the selectins evolved by duplication and rearrangement of individual exons. COS cells transiently expressing the rabbit ELAM-1 cDNA mediate the adhesion of rabbit and human polymorphonuclear leukocytes and are recognized by antibodies prepared against the human protein. Our results suggest that the specificity of molecular interaction between ELAM-1 and its ligand is highly conserved.

Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans.

A large number of diverse cell surface proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. One proposed function for the GPI anchor is that it facilitates the release of the protein from the cell by acting as a target for anchor-specific phospholipases. We and others have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD) (Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem, Biophys. Res. Commun. 150, 476-482; Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984). Because the GPI-PLD recognizes a conserved portion of the anchor, all GPI-anchored proteins are potential substrates for the enzyme. We demonstrate in this communication the production of the plasma GPI-PLD by the islets of Langerhans. GPI-PLD enzymatic activity was found in dog pancreatic microsomes, but not pancreatic juice. Both the pancreatic and plasma enzymes were divalent cation-dependent and had identical substrate specificities. Purified murine islets of Langerhans, as well as alpha and beta cells, contained and released GPI-PLD activity. A GPI-PLD DNA fragment was amplified by polymerase chain reaction from a normal human islet cDNA library; the amplified fragment hybridized with the GPI-PLD cDNA clone. These findings represent the first demonstration of the production of the plasma GPI-PLD by a specific tissue site as well as cell type.

Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D.

A phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) that specifically hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol-glycans (PI-Gs) has recently been purified from human and bovine sera. The primary structure of bovine PI-G PLD has now been determined and the functional activity of the enzyme has been studied. Expression of PI-G PLD complementary DNA in COS cells produced a protein that specifically hydrolyzed the inositol phosphate linkage of the PI-G anchor. Cotransfection of PI-G PLD with a PI-G-anchored protein resulted in the secretion of the PI-G-anchored protein. The results suggest that the expression of PI-G PLD may influence the expression and location of PI-G-anchored proteins.

The use of DNA amplification for genetic counselling related diagnosis in haemophilia B.

A single base pair variation in the coding sequence of coagulation factor IX produces a protein polymorphism detectable with monoclonal antibodies and a restriction fragment length polymorphism (RFLP). This allows carrier and prenatal diagnoses in 48% of Caucasian families segregating for haemophilia B. However, this RFLP cannot be detected by standard Southern blotting, while the antibody assay may give equivocal results in some females and can only allow prenatal diagnoses on second trimester fetal blood samples. We show that, using the polymerase chain reaction, the polymorphic DNA segment can be amplified and directly tested for the presence of the alternative sequences by a non-radioactive procedure that has the advantage of speed (1-2 days), partial automation and applicability to first trimester diagnoses. We also show that the method gives results on a single drop of dried blood.

A factor IX mutation, verified by direct genomic sequencing, causes haemophilia B by a novel mechanism.

A novel factor IX gene mutation (factor IX London 2) has been characterized. This causes severe crm+ haemophilia B as the patient's plasma shows normal factor IX antigen level and less than 1% clotting activity. Sequence analysis of the entire cloned coding and promoter regions revealed a single point mutation: a G----A transition at position 31,119. This region of the patient's DNA was amplified in vitro by the polymerase chain reaction and the nucleotide change was confirmed by direct sequencing of the amplified products. The mutation results in the substitution of the arginine at position 333 by glutamine. This arginine residue is absolutely conserved in the catalytic domain of normal human and bovine factor IX, X and prothrombin. The substitution by glutamine causes the loss of a positive charge from the surface of the factor IX London 2 protein. This mutation pinpoints a previously unknown, functionally critical feature of factor IX which may be involved in substrate or co-factor binding.