Pre-oxygenation using high-flow nasal oxygen vs. tight facemask during rapid sequence induction.

Pre-oxygenation using high-flow nasal oxygen can decrease the risk of desaturation during rapid sequence induction in patients undergoing emergency surgery. Previous studies were single-centre and often in limited settings. This randomised, international, multicentre trial compared high-flow nasal oxygen with standard facemask pre-oxygenation for rapid sequence induction in emergency surgery at all hours of the day and night. A total of 350 adult patients from six centres in Sweden and one in Switzerland undergoing emergency surgery where rapid sequence induction was required were included and randomly allocated to pre-oxygenation with 100% oxygen using high-flow nasal oxygen or a standard tight-fitting facemask. The primary outcome was the number of patients developing oxygen saturations <93% from the start of pre-oxygenation until 1 min after tracheal intubation. Data from 349 of 350 patients who entered the study were analysed (174 in the high-flow nasal oxygen group and 175 in the facemask group). No difference was detected in the number of patients desaturating <93%, five (2.9%) vs. six (3.4%) patients in the high-flow nasal oxygen and facemask group, respectively (p = 0.77). The risk of desaturation was not increased during on-call hours. No difference was seen in end-tidal carbon dioxide levels in the first breath after tracheal intubation or in the number of patients with signs of regurgitation between groups. These results confirm that high-flow nasal oxygen maintains adequate oxygen levels during pre-oxygenation for rapid sequence induction.

DISSEQT-DIStribution-based modeling of SEQuence space Time dynamics.

Rapidly evolving microbes are a challenge to model because of the volatile, complex, and dynamic nature of their populations. We developed the DISSEQT pipeline (DIStribution-based SEQuence space Time dynamics) for analyzing, visualizing, and predicting the evolution of heterogeneous biological populations in multidimensional genetic space, suited for population-based modeling of deep sequencing and high-throughput data. The pipeline is openly available on GitHub (https://github.com/rasmushenningsson/DISSEQT.jl, accessed 23 June 2019) and Synapse (https://www.synapse.org/#!Synapse: syn11425758, accessed 23 June 2019), covering the entire workflow from read alignment to visualization of results. Our pipeline is centered around robust dimension and model reduction algorithms for analysis of genotypic data with additional capabilities for including phenotypic features to explore dynamic genotype-phenotype maps. We illustrate its utility and capacity with examples from evolving RNA virus populations, which present one of the highest degrees of genetic heterogeneity within a given population found in nature. Using our pipeline, we empirically reconstruct the evolutionary trajectories of evolving populations in sequence space and genotype-phenotype fitness landscapes. We show that while sequence space is vastly multidimensional, the relevant genetic space of evolving microbial populations is of intrinsically low dimension. In addition, evolutionary trajectories of these populations can be faithfully monitored to identify the key minority genotypes contributing most to evolution. Finally, we show that empirical fitness landscapes, when reconstructed to include minority variants, can predict phenotype from genotype with high accuracy.

Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia.

We investigated, by a combined in vivo and in vitro approach, the temporal changes of islet nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme oxygenase (HO)-derived carbon monoxide (CO) production in relation to insulin and glucagon secretion during acute endotoxemia induced by lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and cGMP content after in vitro incubation, the insulin response to glucose in vivo and in vitro, and the insulin and glucagon responses to the adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in islet tissue. Immunocytochemistry revealed a marked expression of iNOS in many beta-cells, but only in single alpha-cells after LPS. Moreover, biochemical analysis showed a time dependent and markedly increased production of NO and CO in these islets. Addition of a NOS inhibitor to such islets evoked a marked potentiation of glucose-stimulated insulin release. Finally, after incubation in vitro, a marked suppression of NO production by both exogenous CO and glucagon was observed in control islets. This effect occurred independently of a concomitant inhibition of guanylyl cyclase. We suggest that the impairing effect of increased production of islet NO on insulin secretion during acute endotoxemia is antagonized by increased activities of the islet cAMP and HO-CO systems, constituting important compensatory mechanisms against the noxious and diabetogenic actions of NO in endocrine pancreas.

Total parenteral nutrition modulates hormone release by stimulating expression and activity of inducible nitric oxide synthase in rat pancreatic islets.

The expression and activities of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in relation to insulin and glucagon secretory mechanisms were investigated in islets isolated from rats subjected to total parenteral nutrition (TPN) for 10 d. TPN is known to result in significantly increased levels of plasma lipids during the infusion time. In comparison with islets from freely fed control rats, islets taken from TPN rats at d 10 displayed a marked decrease in glucose-stimulated insulin release (4.65 +/- 0.45 ng/[islet x h] vs 10.25 +/- 0.65 for controls) (p < 0.001) accompanied by a strong iNOS activity (18.3 +/- 1.1 pmol of NO/[min x mg of protein]) and a modestly reduced cNOS activity (11.3 +/- 3.2 pmol of NO/[min x mg of protein] vs 17.7 +/- 1.7 for controls) (p < 0.01). Similarly, Western blots showed the expression of iNOS protein as well as a significant reduction in cNOS protein in islets from TPN-treated rats. The enhanced NO production, which is known to inhibit glucose-stimulated insulin release, was manifested as a strong increase in the cyclic guanosine 5'-monophosphate content in the islets of TPN-treated rats (1586 +/- 40 amol/islet vs 695 +/- 64 [p < 0.001] for controls). Moreover, the content of cyclic adenosine monophosphate (cAMP) was greatly increased in the TPN islets (80.4 +/- 2.1 fmol/islet vs 42.6 +/- 2.6 [p < 0.001] for controls). The decrease in glucose-stimulated insulin release was associated with an increase in the activity of the secretory pathway regulated by the cAMP system in the islets of TPN-treated rats, since the release of insulin stimulated by the phosphodiesterase inhibitor isobutylmethylxanthine was greatly increased both in vivo after iv injection and after in vitro incubation of isolated islets. By contrast, the release of glucagon was clearly reduced in islets taken from TPN-treated rats (33.5 +/- 1.5 pg/[islet x h] vs 45.5 +/- 2.2 for controls) (p < 0.01) when islets were incubated at low glucose (1.0 mmol/L). The data show that long-term TPN treatment in rats brings about impairment of glucose-stimulated insulin release, that might be explained by iNOS expression and a marked iNOS-derived NO production in the beta-cells. The release of glucagon, on the other hand, is probably decreased by a direct "nutrient effect" of the enhanced plasma lipids. The results also suggest that the islets of TPN-treated rats have developed compensatory insulin secretory mechanisms by increasing the activity of their beta-cell cAMP system.

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release.

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.

Islet constitutive nitric oxide synthase and glucose regulation of insulin release in mice.

We have studied, by a combined in vitro and in vivo approach, the relation between the inhibitory action of N(G)-nitro-l-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase (NOS), on the activity of islet constitutive NOS (cNOS) and glucose regulation of islet hormone release in mice. The cNOS activity in islets incubated in vitro at 20 mM glucose was not appreciably affected by 0.05 or 0.5 mM L-NAME, but was greatly suppressed (-60%) by 5 mM L-NAME. Similarly, glucose-stimulated insulin release was unaffected by the lower concentrations of L-NAME but greatly enhanced in the presence of 5 mM of the NOS inhibitor. In incubated islets inhibition of cNOS activity resulted in a modestly enhanced insulin release in the absence of glucose, did not display any effect at physiological or subphysiological glucose concentrations, but resulted in a markedly potentiated insulin release at hyperglycaemic glucose concentrations. In the absence of glucose, glucagon secretion was suppressed by L-NAME. The dynamics of glucose-induced insulin release and (45)Ca(2+) efflux from perifused islets revealed that L-NAME caused an immediate potentiation of insulin release, and a slight increase in (45)Ca(2+) efflux. In islets depolarized with 30 mM K(+) in the presence of the K(+)(ATP) channel opener, diazoxide, L-NAME still greatly potentiated glucose-induced insulin release. Finally, an i.v. injection of glucose to mice pretreated with L-NAME was followed by a markedly potentiated insulin response, and an improved glucose tolerance. In accordance, islets isolated directly ex vivo after L-NAME injection displayed a markedly reduced cNOS activity. In conclusion, we have shown here, for the first time, that biochemically verified suppression of islet cNOS activity, induced by the NOS inhibitor L-NAME, is accompanied by a marked potentiation of glucose-stimulated insulin release both in vitro and in vivo. The major action of NO to inhibit glucose-induced insulin release is probably not primarily linked to changes in Ca(2+) fluxes and is exerted mainly independently of membrane depolarization events.

Morphological evidence for the existence of nitric oxide and carbon monoxide pathways in the rat islets of Langerhans: an immunocytochemical and confocal microscopical study.

AIMS/HYPOTHESIS: To map the cellular location of inducible and constitutive nitric oxide synthase and haem oxygenase in rat islets to clarify the morphological background to putative nitric oxide and carbon monoxide pathways. METHODS: Immunocytochemistry and confocal microscopy. RESULTS: After treatment with endotoxin, immunoreactivity for inducible nitric oxide synthase was expressed in a large number of islet cells, most of which were insulin-immunoreactive beta cells and in single glucagon-immunoreactive and pancreatic polypeptide-immunoreactive cells. Somatostatin-immunoreactive cells lacked immunoreactivity for inducible nitric oxide synthase. In untreated rats, immunoreactivity for constitutive nitric oxide synthase occurred in the majority of insulin-immunoreactive and glucagon-immunoreactive cells, in most pancreatic polypeptide-immunoreactive and somatostatin-immunoreactive cells and in islet nerves. Similarly, immunoreactivity for constitutive haem oxygenase was detected in all four types of islet cells. Endotoxin treatment did not change the pattern of immunoreactivity for constitutive and inducible haem oxygenase. After treatment with alloxan, insulin-immunoreactivity was observed only in single islet cells, being almost devoid of immunoreactivity for constitutive nitric oxide synthase and haem oxygenase. CONCLUSION/INTERPRETATION: In vivo endotoxin-induced expression of inducible nitric oxide synthase in insulin-producing and in scattered glucagon-producing and pancreatic polypeptide-producing cells strengthens previous suggestions of a pathophysiological role for inducible nitric oxide synthase in the development of insulin-dependent diabetes mellitus. The presence of constitutive nitric oxide synthase and haem oxygenase in all four types of islet cells, together with recent functional data of ours support roles for nitric oxide and carbon monoxide as intracellular, paracrine or neurocrine modulators of islet hormone secretion.

Dysfunction of the islet lysosomal system conveys impairment of glucose-induced insulin release in the diabetic GK rat.

Accumulated evidence links an important signal involved in glucose-stimulated insulin release to the activation of the islet lysosomal glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. We have analyzed the function of the lysosomal system/lysosomal enzyme activities in pancreatic islets of young (6-8 weeks), spontaneously diabetic, GK (Goto-Kakizaki) rats and Wistar control rats in relation to glucose-induced insulin release. The insulin secretory response to glucose was markedly impaired in the GK rat, but was restored by the adenylate cyclase activator forskolin. Islet activities of classical lysosomal enzymes, e.g.. acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and cathepsin D, were reduced by 20-35% in the GK rat compared with those in Wistar controls. In contrast, the activities of the lysosomal alpha-glucosidehydrolases, i.e.. acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, were increased by 40-50%. Neutral alpha-glucosidase (endoplasmic reticulum) was unaffected. Comparative analysis of liver tissue showed that lysosomal enzyme activities were of the same magnitude in GK and Wistar rats. Notably, in Wistar rats, the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase were approximately 15-fold higher in islets than in liver. Other lysosomal enzymes did not display such a difference. Normalization of glycemia in GK rats by phlorizin administered for 9 days did not influence either the lysosomal alpha-glucosidehydrolase activities or other lysosomal enzyme activities in GK islets. Finally, the pseudotetrasaccharide acarbose, which accumulates in the lysosomal system, inhibited acid glucan-1,4-alpha-glucosidase activity in parallel with its inhibitory action on glucose-induced insulin release in intact Wistar islets, whereas no effect was recorded for either parameter in intact GK islets. In contrast, acarbose inhibited the enzyme activity equally in islet homogenates from both GK and Wistar rats, showing that the catalytic activity of the enzyme itself in disrupted cells was unaffected. We propose that dysfunction of the islet lysosomal/vacuolar system is an important defect impairing the transduction mechanisms for glucose-induced insulin release in the GK rat.

Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release: a biochemical, immunohistochemical, and confocal microscopic study.

Carbon monoxide (CO) has been suggested as a novel messenger molecule in the brain. We now report on the cellular localization and hormone secretory function of a CO-producing constitutive heme oxygenase (HO-2) in mouse islets. Islet homogenates produced large amounts of CO which were suppressed dose-dependently by the HO inhibitor zincprotoporphyrin-IX (ZnPP-IX). We also show, for the first time, that glucose markedly stimulates the HO activity (CO production) in intact islets. A further potentiation was induced by the HO substrate hemin. Western blot showed that islet tissue expressed HO-2, and confocal microscopy revealed that HO-2 resided in insulin, glucagon, somatostatin, and pancreatic polypeptide cells. ZnPP-IX dose-dependently inhibited, whereas hemin enhanced, both insulin and glucagon secretion from glucose-stimulated islets. Stimulation or inhibition of CO production was accompanied by corresponding changes in islet cGMP levels. Exogenously applied CO stimulated insulin and glucagon release from isolated islets, whereas exogenous nitric oxide (NO) inhibited insulin and stimulated glucagon release. Islets stimulated by glucose or L-arginine displayed a marked increase in their NO-synthase (NOS) activity. Such an increase was suppressed by hemin, conceivably because NOS activity was inhibited by hemin-derived CO. Consequently, hemin enhanced L-arginine-induced insulin secretion. Insulin release stimulated by either hemin-derived CO or exogenous CO was strongly inhibited by the guanylate cyclase inhibitor ODQ, but it was unaffected by ZnPP-IX. Glucagon release induced by CO (but not by hemin) was inhibited by ODQ and partly inhibited by ZnPP-IX. We propose that the islets of Langerhans are equipped with a heme oxygenase-carbon monoxide pathway, which constitutes a novel regulatory system of physiological importance for the stimulation of insulin and glucagon release. This pathway is stimulated by glucose, is at least partly dependent on the cGMP system, and displays interaction with islet NOS activity.

Arginine-induced insulin release is decreased and glucagon increased in parallel with islet NO production.

Nitric oxide (NO) produced by islet constitutive NO synthase (cNOS) is a putative modulator of islet hormone secretion. We show here for the first time that the release of insulin induced by L-arginine or L-homoarginine is inhibited and that of glucagon stimulated in parallel with the rate of islet NO production. It was found that L-homoarginine was approximately 25-30% less potent than L-arginine as an insulin secretagogue but equally potent as a glucagon secretagogue. Biochemical determination of islet cNOS activity revealed that the NO production with L-homoarginine as substrate was only approximately 40% of that of L-arginine. Selective inhibition of islet cNOS potentiated insulin release during amino acid stimulation. Moreover, inhibition of cNOS suppressed glucagon release, more so with L-arginine than with L-homoarginine as secretagogue, reflecting the relative rates of their NO production. In K+-depolarized islets, inhibition of cNOS enhanced the insulin response to L-arginine by 50% and that to L-homoarginine by 23%, largely corresponding to their relative NO production. The intracellular NO donor hydroxylamine dose dependently inhibited insulin but increased glucagon secretion in K+-depolarized and amino acid-stimulated islets. We conclude that both amino acids have a dual action on insulin release, since their stimulatory effects are reduced in parallel with the rates of their NO production. Furthermore, the greater NO production induced by L-arginine relative to L-homoarginine corresponds to NO-mediated increases in glucagon release. These NO effects are mainly exerted independently of membrane depolarization events.

Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets.

Recent observations suggest that carbon monoxide (CO) may serve as a neuroendocrine modulator in hypothalamus. Here we provide evidence, for the first time, that the islets of Langerhans contain the constitutive isoform of the CO-producing enzyme heme oxygenase (HO-2), the activity of which was found to modulate islet hormone release. Most insulin and glucagon cells in the rat endocrine pancreas expressed strong immunoreactivity for HO-2. In the exocrine parenchyma, scattered HO-2-positive ganglionic cell bodies were occasionally observed. Furthermore, Western blot analysis revealed the presence of HO-2 in isolated islets but not in acinar cells. Islet homogenates displayed a comparatively high HO-2 enzymatic activity measured as CO formation (approximately 600 pmol CO.min-1.mg islet protein-1). This HO-2 enzymatic activity was greatly suppressed by zincprotoporphyrin-IX (ZnPP-IX), a recognized inhibitor of HO activity. Neither ZnPP-IX nor the HO activator, hemin, influenced basal insulin release from isolated rat islets at low (1 mM) glucose. However, glucagon release at 1 mM glucose was increased by hemin and inhibited by ZnPP-IX. The hemin-induced increase in glucagon secretion was abolished by ZnPP-IX. Furthermore, a series of experiments at high glucose (16.7 mM) revealed that hemin induced a dose-dependent potentiation of glucose-stimulated insulin release. Moreover, glucose-induced insulin release was dose-dependently suppressed by ZnPP-IX but unaffected by protoporphyrin-IX, a compound known not to influence HO-2 activity in other tissues. Similarly, glucagon release at high glucose was dose-dependently increased by hemin and suppressed by ZnPP-IX. Finally, the hemin-induced increase in islet hormone release at high glucose was totally abolished by ZnPP-IX. The data strongly suggest that CO production positively modulates both glucagon and insulin secretion. We propose that CO may serve as a novel messenger molecule within the islets of Langerhans.

Patterns of antimicrobial therapy for pediatric patients.

The pattern of antimicrobial prescribing for outpatients and inpatients in a Swedish pediatric department was evaluated in 1975 and 1982. The changes in the pattern during these 7 yr were small and, with regard to outpatients, very similar to those in the whole of Sweden. In these children phenoxymethylpenicillin (penicillin V) was the predominant antimicrobial (approximately 70%), followed by erythromycin and medium wide-spectrum penicillins (ampicillin/amoxycillin). Prescriptions for erythromycin in outpatients increased from 8 to 16%. The corresponding figures for Sweden as a whole were from 13 to 20%. This increase might partly be explained by the higher frequency of pertussis in Sweden in the last few years, but it is probably also a manifestation of an insufficiently motivated widening of the range of indications for erythromycin. A comparison between the prescribing patterns in Sweden and North America showed an obvious difference. In the USA the medium wide-spectrum penicillins are more used than penicillin V. This difference is discussed and found to be probably due to a real difference in antimicrobial policy. Penicillin V and G also dominated the inpatient prescriptions (51% in 1975 and 42% in 1982), which also differed from some North American hospitals.