What is the potential benefit of pre-hospital extracorporeal cardiopulmonary resuscitation for patients with an out-of-hospital cardiac arrest? A predictive modelling study.

AIM: In this predictive modelling study we aimed to investigate how many patients with an out-of-hospital cardiac arrest (OHCA) would benefit from pre-hospital as opposed to in-hospital initiation of extracorporeal cardiopulmonary resuscitation (ECPR). METHODS: A temporal spatial analysis of Utstein data was performed for all adult patients with a non-traumatic OHCA attended by three emergency medical services (EMS) covering the north of the Netherlands during a one-year period. Patients were considered potentially eligible for ECPR if they had a witnessed arrest with immediate bystander CPR, an initial shockable rhythm (or signs of life during resuscitation) and could be presented in an ECPR-centre within 45 minutes of the arrest. Endpoint of interest was defined as the hypothetical number of ECPR eligible patients after 10, 15 and 20 minutes of conventional CPR and upon (hypothetical) arrival in an ECPR-centre as a fraction of the total number of OHCA patients attended by EMS. RESULTS: During the study period 622 OHCA patients were attended, of which 200 (32%) met ECPR eligibility criteria upon EMS arrival. The optimal transition point between conventional CPR and ECPR was found to be after 15 minutes. Hypothetical intra-arrest transport of all patients in whom no return of spontaneous circulation (ROSC) was obtained after that point (n = 84) would have yielded 16/622 (2.5%) patients being potentially ECPR eligible upon hospital arrival (average low-flow time 52 minutes), whereas on-scene initiation of ECPR would have resulted in 84/622 (13.5%) potential candidates (average estimated low-flow time 24 minutes before cannulation). CONCLUSION: Even in healthcare systems with relatively short transport distances to hospital, consideration should be given to pre-hospital initiation of ECPR for OHCA as it shortens low-flow time and increases the number of potentially eligible patients.

Could we use the comfort score instead of the numeric rating pain score? A proof of concept pilot study.

INTRODUCTION: Asking patients about pain in the Emergency Department (ED) when deriving a pain score may aggravate perception of pain due to the nocebo-effect. A strategy for diminishing this nocebo-effect is cognitive reframing. Cognitive reframing of the frequently used pain score (PS) in the ED could theoretically be obtained by using the comfort score (CS). The aim of this study was to evaluate whether or not the CS and PS are interchangeable and therefore, whether or not the CS could safely be used in ED patients. METHODS: In this prospective pilot study we enrolled patients with pain visiting the ED. Participants were asked for both PS and CS in randomized order. CS were inverted (ICS) and compared with PS using the using the Wilcoxon signed rank test. Secondarily we evaluated for patient score preference. RESULTS: In total 100 patients were enrolled. The median PS in these participants was 6 (IQR 4-7) and median ICS was 5 (IQR 3-6). In total, 15 (15%) of the PS and ICS were identical Medians did not differ significantly (p = .115). In 33% of the participants the total difference between the PS and ICS was >2. Participants preferred to be asked for PS over CS (43 vs 15%, p < .00). CONCLUSION: This proof of concept study suggest interchangeability of the PS and the ICS in patients with pain in the ED. However, while not statistically significant, 33% of the patients had a possible clinical significant difference in score outcome, potentially over- or underestimating the patients pain. Whether or not this can be used as a tool for cognitive reframing to reduce perception of pain and medication consumption has yet to be studied.

IGF-I and retinoic acid regulate the distribution pattern of IGFBPs synthesized by the canine mammary tumor cell line CMT-U335.

Stromal-epithelial interactions modulate growth and development in normal and neoplastic mammary gland. The release of IGF binding proteins (IGFBPs) by the stromal compartment of the mammary gland may play a modulating role in the IGF-mediated proliferation of mammary epithelium. Therefore, the IGFBP-expression pattern of the canine mammary tumor cell line U335 (CMT-U335), which has a mesenchymal phenotype, was determined. In addition, the effects of IGFs and all trans retinoic acid (RA) on DNA synthesis, and IGFBP secretion and distribution were examined. The IGFBPs secreted by CMT-U335 were characterized as IGFBP-2, -4, -5, and -6. Moreover, CMT-U335 appeared to be a suitable mammary mesenchymal cell line for study of the regulatory factors of IGFBP expression and the mechanism(s) involved. IGFs and RA enhanced IGFBP concentrations in cell-conditioned medium with IGF-I and RA having an additive effect. The IGF-I-stimulated DNA synthesis, however, was inhibited by RA. The difference between IGF-I and RA was an enhanced IGFBP-5 binding to the extracellular matrix (ECM) by RA, whereas IGF-I reduced binding to the ECM. Because high doses of insulin had no significant effects on IGFBP concentrations in the medium, it is concluded that IGF-I-induced changes in IGFBP concentrations are not mediated by type-IIGF receptors and may be the consequence of IGFBP redistribution.

Predicted primary and antigenic structure of canine corticotropin releasing hormone.

Although the dog has been recognized as a useful model for the study of the cerebrospinal and peripheral actions of corticotropin releasing hormone (CRH) the exact amino acid composition of canine CRH is still unknown. In the present study the structure of canine CRH was predicted from the partial sequence of the gene encoding canine CRH. The CRH gene was amplified from genomic DNA obtained from white blood cells by a polymerase chain reaction and subsequently sequenced using the dideoxy method. The likely structure of canine CRH is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-NH2, which is identical to the structure of human, rat and equine CRH. PEPSCAN analysis of 3 different CRH antisera predicted an antiserum raised against a conjugate of human CRH and CNBr -activated thyroglobulin to be the antiserum of choice for the measurement of CRH in the dog. Preliminary data confirmed the existence of the highest cross-reactivity of a canine hypothalamus extract, known to have a high content of CRH, with antisera directed against human, rat CRH. As a result of the present study immunological tools for CRH estimations are characterized. Also, a homologous DNA probe for in situ hybridizations has become available for further investigations.

Elucidation of the sequence of canine (pro)-calcitonin. A molecular biological and protein chemical approach.

From the canine thyroid gland a calcitonin (CT) immunoreactive peptide was purified by successive aqueous acid acetone extraction, gel filtration and HPLC. Gas-phase sequencing of the purified peptide showed that the first 25 amino acids had 65% sequence homology with the amino-terminus of the human CT prohormone. A canine cDNA library was then made from the thyroid gland. A plasmid was isolated containing a sequence that is homologous to part of exon 3, and the complete sequence of exon 4 of the human mRNA encoding preproCT. From this cDNA the amino acid sequence of canine CT is predicted. In comparison with well-known CT sequences of other species, the strongest homology exists with bovine, porcine and ovine CT.

The gene encoding proopiomelanocortin in the dog.

The regulation of the synthesis of ACTH in the dog is of interest for studies of the physiology of the pituitary-adrenocortical axis as well as for studies of the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite this broad interest the nucleotide sequence encoding ACTH and its precursor proopiomelanocortin (POMC) is not known, nor is it clear whether there are differences in POMC mRNA from the anterior lobe or the intermediate lobe of the normal pituitary or from pituitary tumours causing ACTH excess. Following the preparation of a cDNA library from the canine intermediate lobe of the pituitary gland, the part of the mRNA that is translated into the proopiomelanocortin prohormone was amplified using a polymerase chain reaction. Sequence analysis revealed the highest homology with the porcine mRNA sequence. Translation in a single reading frame revealed highly homologous areas in the amino-terminal, carboxy-terminal, and ACTH part of the prohormone, whereas a high diversity was noticed at the sequence preceding ACTH and the beginning of beta-lipotropin. Northern blot analysis disclosed the presence of a POMC mRNA of approximately 1300 nucleotides. There were no size differences between the anterior lobe, intermediate lobe, and pituitary tumour derived POMC mRNA. The highest expression levels of POMC mRNA as related to the expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase were found in the intermediate lobe of the canine pituitary gland. It is concluded that excessive production of ACTH by pituitary tumours is not caused by relatively high expression levels or alterations in the size of mRNA.

Comparison of different methods of measuring protein and albumin in pigeon sera.

Columbine serum total protein (TP) and albumin concentrations were determined using the biuret method and the bromocresol green dye binding (BCG) method or serum protein electrophoresis on cellulose acetate membranes (SPE). Results obtained using human and pigeon standards were compared. When pigeon albumin was used as a standard. TP values were consistently higher compared with values obtained using human protein as a standard. However, there was a high correlation between the results obtained with the two standards. The correlation between the BCG method and SPE for serum albumin determination was poor, irrespective of the standard used. The method cannot be recommended for pigeon blood. For avian clinical practice it is advised to establish TP concentration using the biuret method and a human standard and to calculate albumin concentration from the results of TP and SPE.

Effects of bromocriptine on corticotrophin, melanotrophin and corticosteroid secretion in dogs with pituitary-dependent hyperadrenocorticism.

Dogs with spontaneous pituitary-dependent hyperadrenocorticism were divided into two groups, one with normal plasma concentrations of alpha-MSH (normal alpha-MSH dogs, n = 26) and the other with high plasma concentrations of alpha-MSH (high alpha-MSH dogs, n = 14), on the presumption that high alpha-MSH concentrations indicated a parent cell of pars intermedia origin. The urinary corticoid/creatinine ratios of the high alpha-MSH dogs were significantly higher than those of the normal alpha-MSH dogs. The percentage decrease of the corticoid/creatinine ratios following dexamethasone administration was significantly higher in the normal alpha-MSH dogs than in the high alpha-MSH dogs. Dexamethasone resistance occurred in both the normal alpha-MSH dogs (4 out of 26) and the high alpha-MSH dogs (7 out of 14), indicating a relative rather than an absolute difference. The short-term effect of orally administered bromocriptine, at a dose (10 micrograms/kg body weight) known to be effective in lowering prolactin concentrations in dogs, was investigated by measuring concentrations of cortisol, ACTH and alpha-MSH in plasma at 4, 6 and 8 h after administration. Significant decreases were observed for cortisol in both groups and for alpha-MSH only in the high alpha-MSH dogs. The effect of 5 days of bromocriptine administration (10 micrograms at 12-h intervals) was assessed by measurements of urinary corticoid/creatinine ratios. Considering both groups as a whole, only the corticoid/creatinine ratios of the high alpha-MSH dogs decreased significantly on the first day of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells.

Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, ribonuclease-sensitive fraction from the cytosol of uninfected cells. This fraction stimulated the initiation about 3-fold and the replication of origin fragments 5-10-fold. Sedimentation analysis indicated the presence of a fast-sedimenting and a slow-sedimenting component which complemented each other. At least part of the stimulation was caused by low-molecular-mass RNA.

The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.