Partnering with survivors & families to determine research priorities for adult out-of-hospital cardiac arrest: A James Lind Alliance Priority Setting Partnership.

BACKGROUND: Research priority setting in health care has historically been done by expert health care providers and researchers and has not involved patients, family or the public. Survivors & family members have been particularly absent from this process in the field of resuscitation research and specifically adult out of hospital cardiac arrest (OHCA). As such, we sought to conduct a priority setting exercise in partnership with survivors, lay responders and their families in order to ensure that their priorities were visible. We partnered with the James Lind Alliance (UK) and used their commonly used consensus methodology for Public Priority Setting Partnerships (PSPs) to identify research priorities that reflected the perspectives of all stakeholders. METHODS: We used two rounds of public and health care professional surveys to create the initial priority lists. The initial survey collected open-ended questions while the second round consolidated the list of initial questions into a refined list for prioritization. This was done by reviewing existing evidence and thematic categorization by the multi-disciplinary steering committee. An in-person consensus workshop was conducted to come to consensus on the top ten priorities from all perspectives. The McMaster PPEET tool was used to measure engagement. RESULTS: The initial survey yielded more than 425 responses and 1450 "questions" from survivors and family members (18%), lay responders, health care providers and others. The second survey asked participants to rank a short list of 125 questions. The final top 25 questions were brought to the in-person meeting, and a top ten were selected through the JLA consensus process. The final list of top ten questions included how to improve the rate of lay responder CPR, what interventions used at the scene of an arrest can improve resuscitation and survival, how survival can be improved in rural areas of Canada, what resuscitation medications are most effective, what care patient's family members need, what post-discharge support is needed for survivors, how communication should work for everyone involved with a cardiac arrest, what factors best predict neurologically intact survival, whether biomarkers/genetic tests are effective in predicting OHCA and more research on the short and long-term psycho-social impacts of OHCA on survivors. The PPEET showed overwhelmingly positive results for the patient and family engagement experience during the final workshop. CONCLUSIONS: This inclusive research priority setting provides essential information for those doing resuscitation research internationally. The results provide a guide for priority areas of research and should drive our community to focus on questions that matter to survivors and their families in our work. In particular the Canadian Resuscitation Outcomes Consortium will be incorporating the top ten list into its strategic plan for the future.

Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A::growth hormone releasing factor) produced in Escherichia coli.

DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.

RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity.

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.

Human interleukin 1 beta fused to the human growth hormone signal peptide is N-glycosylated and secreted by Chinese hamster ovary cells.

A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.

Expression vector promoting the synthesis and export of the human growth-hormone-releasing factor in Escherichia coli.

We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein. A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF). The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space. Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.

Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli.

To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Synthetic oligodeoxynucleotides, either unmodified or linked to an intercalating agent, have been used to prevent cDNA elongation by the AMV reverse transcriptase. Oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA part bound to the antisense oligonucleotide by the RNase-H activity associated with the retroviral polymerase.

Molecular basis for the different binding properties of benzodiazepines to human and bovine peripheral-type benzodiazepine receptors.

The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro5-4864, and isoquinoline carboxamides such as PK11195. These two compounds are reversible competitive inhibitors of each other. However, while the binding affinity of Ro5-4864 varies enormously across species, PK11195 always displays high affinity, suggesting that their binding domains are overlapping but not identical. We report here that recombinant human and bovine PBR produced in yeast, a microorganism devoid of endogenous PBR, can be labelled with [3H]PK11195, but only the human receptor can be labelled with [3H]Ro5-4864. Furthermore, we identified, through the binding analysis of human-bovine chimaeric receptors, a region near the C-terminal end of the PBR, with only five non-conserved amino acids between human and bovine sequences, as responsible for the difference in high affinity binding of Ro5-4864 to the two receptors.

SR141716A, a potent and selective antagonist of the brain cannabinoid receptor.

SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.

Antisense effect of oligodeoxynucleotides complementary to the mini-exon sequence of the protozoan parasite Leishmania amazonensis.

We have targeted the mini-exon of Leishmania amazonensis, the sequence present at the 5' end of every mRNA of this protozoan parasite, with a complementary 12-mer, either unmodified (12 Le II) or linked to an acridine derivative (12 Le II Acr). Physical measurements performed either in solution or on nitrocellulose filters showed that the two oligomers exhibited the same affinity for both DNA and RNA target sequences. Furthermore, the two oligomers 12 Le II and 12 Le II Acr inhibited in vitro translation of L amazonensis mRNAs, in a wheat germ extract, to the same extent. Those results indicated that the intercalating agent did not stabilize the duplex formed by the antisense oligomer and its target sequence.

The maximum rate of gene expression is dependent on the downstream context of unfavourable codons.

Presented here is an experimental demonstration of our theoretical predictions on the role of the downstream context of unfavourable codons in a gene on its expression level. Six non clustered AGG codons were inserted in the chloramphenicol acetyltransferase (cat) gene of E. coli and the expression of this modified gene (cat4) was compared with that of a cat gene in which four clustered AGG codons were inserted (cat2 gene). As predicted, the rate of production of the corresponding CAT4 and CAT2 proteins is equal as long as the rate of transcription of the gene does not exceed a given limit. When this limit is exceeded, production of CAT4 continues to increase, whereas CAT2 production decreases dramatically. Various consequences and possible applications of this downstream context effect are discussed.

Colicin A lysis protein promotes extracellular release of active human growth hormone accumulated in Escherichia coli cytoplasm.

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction. The results indicate that Cal causes the excretion of non-aggregated Met-hGH from the cytoplasm to the culture medium and that the Met-hGH is correctly folded since the released Met-hGH is antigenically indistinguishable from the authentic mature hGH and is biologically active in binding to specific receptor sites.

Evidence for the presence of beta 3-adrenergic receptor mRNA in the human brain.

The beta 3-adrenergic receptor (AR) is widely distributed in peripheral tissues, but up to now it has not been detected in the central nervous system. By using the polymerase chain reaction (PCR) technique, we found the beta 3-AR mRNA to be present in all the regions of the human brain we investigated. The quantities found were very low compared to those of the beta 1-AR and beta 2-AR mRNAs, being hardly detectable in adult brain. In contrast, the brain of very young infants contained about 100 times more beta 3-AR mRNA than the adult brain, whereas the amounts of beta 1-AR and beta 2-AR transcripts were essentially the same. In addition, using PCR we have cloned a central beta 3-AR coding region from a human frontal cortex cDNA library and have found it to be identical to the corresponding peripheral sequence.

Effect of serotonin on cytokine mRNA expression in rat hippocampal astrocytes.

Serotonin is a widely distributed neurotransmitter which elicits a range of central activities. We examined the effect of serotonin on cytokine mRNA expression by rat hippocampal astrocytes in primary cultures. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that interleukin-6 (IL6) mRNA is expressed after 10(-12) M serotonin stimulation whereas transforming growth factor-beta (TGF beta) and tumor necrosis factor (TNF alpha) are induced by 10(-10) M serotonin. These inductions appeared after 1 h stimulation for IL6 and TNF alpha, whereas that of TGF beta appeared after 4 h. The present results provide the first evidence that serotonin can influence astrocyte cytokine production, and thus this neurotransmitter may be considered a potential neuroimmunomodulator.

The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK(1) receptor.

1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK(1) receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK(1) receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [3H]SR 27897 binding properties, Lys(115), Lys(187), Phe(198), Trp(209), Leu(214) and Asn(333). In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe(97), Gly(122), Phe(198), Trp(209), Ile(229), Asn(333), Arg(336) and Leu(356) or increased it, Tyr(48), Cys(94), Asn(98), Leu(217) and Ser(359). Only mutations of Phe(198), Trp(209) and Asn(333) affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK(1) receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.

Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S.

We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.

Contrasting roles of leu(356) in the human CCK(1) receptor for antagonist SR 27897 and agonist SR 146131 binding.

A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK(1)), SR 146131 (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From binding and activity assays with chimeric constructs of human CCK(1) and the cholecystokinin subtype 2 receptor (CCK(2)) and receptors carrying point mutations, we show that Leu(356), situated in transmembrane domain seven in the CCK(1) receptor, is a putative contact point for SR 146131. In contrast, Leu(356) is probably not in contact with the CCK(1) receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK(1) receptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK(2) receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK(2) receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK(1) receptor than in the CCK(2) receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.

Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB(2) receptor.

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2. 1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)- pyrazo le-3-carboxamide) on the human cannabinoid CB(2) receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser(161) and Ser(165), were singly mutated to the cognate cannabinoid CB(1) receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB(2) receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB(2) receptor-specific residue, Cys(175) resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.

Up-regulation of 5-HT2 receptors in the rat brain by repeated administration of SR 46349B, a selective 5-HT2 receptor antagonist.

Chronic administration (twice a day for three days and on the morning of the fourth day) of SR 46349B (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3-(2-fluoroph enyl)propen-1- yl]phenol hemifumarate) (10 mg/kg, orally), a selective 5-HT2 receptor antagonist, caused 24 h later a marked increase (+42%) of the maximum binding capacity of [3H]ketanserin in rat brain cortical membranes without change in its affinity constant. Further, administration of the 5-HT2 receptor agonist, (+/-)-DOI((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (1 mg/kg, i.p.), produced in chronic SR 46349B treated rats a significant increase in the amount of [3H]-inositol phosphate compared to corresponding controls. In addition, subacute administration of SR 46349B caused a 2-fold increase in the head-twitch response to (+/-)-DOI (0.5 mg/kg, i.p.). This enhanced response was blocked by an acute administration of ritanserin (6-(2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-7- methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) (10 mg/kg). Finally, a significant enhancement (+29%) of 5-HT2 receptor mRNA levels was observed in the cortex. Taken together, these data showed that an up-regulation of 5-HT2 receptors occurred in rats following repeated treatment with a selective 5-HT2 receptor antagonist. The effects of SR 46349B on 5-HT2 receptors might implicate pre-translational regulation.