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Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)–like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P < .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P < .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)–like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P< .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P< .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P≤ .001) and tissue damage by 25% (P≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Objective:: Neonates with necrotizing enterocolitis (NEC) have higher calprotectin levels in stool than do healthy neonates. However, it is not known whether high stool calprotectin at the onset of bowel symptoms identifies neonates who truly have NEC vs other bowel disorders. Study Design:: Neonates were eligible for this study when an x-ray was ordered to 'rule-out NEC'. Stool calprotectin was quantified at that time and in a follow-up stool. Each episode was later categorized as NEC or not NEC. The location of calprotectin in the bowel was determined by immunohistochemistry. Results:: Neonates with NEC had higher initial and follow-up stool calprotectin levels than did neonates without NEC. Calprotectin in bowel from neonates with NEC was within neutrophil extracellular traps (NETs). Conclusion:: At the onset of signs concerning for NEC, fecal calprotectin is likely to be higher in neonates with NEC. Calprotectin in their stools is exported from neutrophils via NETs.

Over the past several years, less invasive methods of providing ventilatory support have arisen to help support patients with respiratory insufficiency and airway obstruction. The application of noninvasive ventilation (NIV) in these situations can provide short- to medium-term aid for certain patient populations without introducing the risks associated with endotracheal intubation. In this article, we review the basics of NIV, discuss the accepted indications for NIV in the ICU, explore the perioperative indications for NIV, including use in patients with obstructive sleep apnea, and finally, provide practical tips for implementing NIV in the ICU and anesthesia recovery areas.

The timing of performing a tracheostomy in patients needing prolonged mechanical ventilation has been the subject of many studies over the years. This review surveys the published literature on this topic and describes the findings from the most recent multicenter randomized trials. These data are then used to form the most up-to-date conclusions regarding best practices to guide decisions on timing of tracheostomy in ventilated ICU patients.

Interleukin-2 (IL-2) is a cytokine that produces an in vivo immunostimulating effect when given systemically. This action may arise from stimulating the growth and expansion of cytotoxic T cells that in turn activate natural killer cells to attack tumors such as malignant melanoma and renal cell carcinoma. Therefore, administration of high-dose IL-2 can invoke an antitumor response in some patients. However, high-dose IL-2 induces severe and sometimes life-threatening toxicity, including vascular leak syndrome, hypotension, fever, and end-organ damage. Renal, hepatic, pulmonary, cardiac, and neurologic side effects have been described. Patients receiving IL-2 therefore require close monitoring to detect and treat life-threatening complications arising from treatment. The authors describe the steps taken to begin an IL-2 biotherapy program in a small ICU at a regional comprehensive cancer center. The addition of “elective” IL-2 admissions to the ICU has had a beneficial effect on stabilizing the daily census. In addition, it has brought a new cohort of patients who are highly appreciative of the care they receive and provides the nursing staff a new opportunity to develop expertise with cancer biotherapy.

Blockade of voltage-gated sodium (Na+) channels by local anesthetics represents the main mechanism for inhibition of impulse propagation. Local anesthetic-induced potassium (K+) channel inhibition is also known to influence transmission of sensory impulses and to potentiate inhibition. K+channels involved in this mechanism may belong to the emerging family of background tandem pore domain K+channels (2P K+channels). To determine more precisely the effects of local anesthetics on members of this ion channel family, we heterologously expressed the 2P K+channels TASK-2 (KCNK5), TASK-1 (KCNK3), and chimeric TASK-1/TASK-2 channels in oocytes of Xenopus laevis. TASK-2 cDNA-transfected HEK 293 cells were used for single-channel recordings. Local anesthetic inhibition of TASK-2 was dose-dependent, agent-specific, and stereoselective. The IC50values for R-(+)-bupivacaine and S-(–)-bupivacaine were 17 and 43 μM and for R-(+)-ropivacaine and S-(–)-ropivacaine, 85 and 236 μM. Lidocaine (1 mM) inhibited TASK-2 currents by 55 ± 4%, whereas its quaternary positively charged analog N-ethyl lidocaine (QX314) had no effect. Bupivacaine (100 μM) decreased channel open probability from 20.8 ± 1.6% to 5.6 ± 2.2%. Local anesthetics [300 μM R-(+)-bupivacaine] caused significantly greater depolarization of the resting membrane potential of TASK-2-expressing oocytes compared with water-injected control oocytes (15.8 ± 2.5 mV versus 0.1 ± 0.05 mV; p< 0.001). Chimeric TASK-1/TASK-2 2P K+channel subunits that retained pH sensitivity demonstrated that the carboxy domain of TASK-2 mediates the greater local anesthetic sensitivity of TASK-2. These results show that clinically achievable concentrations of local anesthetics inhibit background K+channel function and may thereby enhance conduction blockade.

The volatile anesthetics are widely used in clinical practice today to produce a state of general anesthesia. But despite more than 150 years of use and substantial scientific investigation, the mechanism by which they produce central nervous system depression remains elusive. Complete understanding of the cellular and molecular basis of the anesthetized state produced by volatile anesthetics most likely involves modulation of the activity of ion channel proteins, these macromolecules provide the most likely molecular targets for these agents. Many studies suggest the involvement of GABAergic and glutamatergic receptor systems in mediating the action of volatile anesthetics. Another ionic current found ubiquitously in neuronal tissues, background potassium currents (also known as resting or leak K+ currents), have recently emerged as plausible targets for volatile anesthetics. A unique structural class of K+ channels with two pore-forming sequences in tandem (2P K+ channels) contributes significantly to background K+ currents. The complete identification of all the 2P K+ channel family members has likely been accomplished. Within intact neuronal systems, background K+ channels are responsible for essential inhibition, these actions are enhanced by volatile anesthetics. Thus, members of this family have emerged as strong candidates for the molecular site of volatile anesthetic action.

Despite over 150 years of clinical use, the mechanism and molecular elements by which volatile anesthetics produce unconsciousness are not established. Although enhanced activity of inhibitory neurotransmitter systems (GABAA) and depression of excitatory neurotransmitter systems (NMDA) probably contribute to the anesthetic state, the role of other ion channels families have also been studied. Potassium channels represent the largest group of mammalian ion channels and their activity to reduce neuronal excitability makes them viable candidates as sites of anesthetic action. Several studies from the 1970s and 80s identified volatile anesthetic enhancement of neuronal potassium currents. More recently, a new family of K channels with a unique structure (tandem pore domains) that may be responsible for baseline or background K currents have been isolated and some members of this family can be activated by volatile anesthetics. These emerging findings suggest a new molecular mechanism by which volatile anesthetics may mediate central nervous system depression.

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.

1.The effects of three barbiturates and the local anesthetic procaine on the ion channel function of mouse nicotinic acetylcholine receptor (nAChR) muscle subtype expressed inXenopus laevis oocytes were examined by whole-cell voltage-clamp technique.2.A concentration-response curve for the specific nicotinic agonist dimethylphenylpiperazinium iodide (DMPP) was first determined. This agonist produced increasing whole-cell currents up to a concentration of 100µM (EC50 = 13µM), then decreased responses at higher concentrations.3.The barbiturates (amobarbital, secobarbital, pentobarbital) and procaine produced reversible inhibition of DMPP-induced currents at clinically used concentrations. The two classes of drugs differed in the voltage dependence of the inhibition: procaine-induced inhibition was increased at more negative transmembrane holding potentials (-90 vs. -45 mV); whereas amobarbital-induced inhibition did not vary at different transmembrane potentials.4.Mutant forms of the nAChR, containing single amino acid changes in the M2 regions ofa andß subunits, showed increased sensitivity to procaine but no change in sensitivity to amobarbital-induced inhibition.5.These electrophysiologic studies provide further evidence that barbiturates and local anesthetics produce inhibition of the nAChR at different sites.

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the beta-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

The lethal spotted (ls) mouse has been used as a model for the human disorder Hirschsprung's disease, because as in the latter condition, ls/ls homozygotes are born without ganglion cells in their terminal colons and, without surgical intervention, die early as a consequence of intestinal obstruction. Previous studies have led to the conclusion that hereditary aganglionosis in ls/ls mice occurs because neural crest-derived enteric neuroblasts fail to colonize the distal large intestine during embryogenesis, perhaps due to a primary defect in non-neuroblastic mesenchyme rather than migrating neuroblasts themselves. In this investigation, the latter issue was addressed directly, in vivo, by comparing the distributions of ls/ls and wild-type neurons in aggregation chimeras. Expression of a transgene, D beta H-nlacZ, in enteric neurons derived from the vagal neural crest, was used as a marker for ls/ls enteric neurons in chimeric mice. In these animals, when greater than 20% of the cells were wild-type, the ls/ls phenotype was rescued; such mice were neither spotted nor aganglionic. In addition, these 'rescued' mice had mixtures of ls/ls and wild-type neurons throughout their gastrointestinal systems including distal rectum. In contrast, mice with smaller relative numbers of wild-type cells exhibited the classic ls/ls phenotype. The aganglionic terminal bowel of the latter mice contained neither ls/ls nor wild-type neurons. These results confirm that the primary defect in ls/ls embryos is not autonomous to enteric neuroblasts, but instead exists in the non-neuroblastic mesenchyme of the large intestine.

The dopamine beta-hydroxylase promoter has been shown to direct expression of the reporter gene product, beta-galactosidase, to enteric neurons and putative embryonic neuroblasts in transgenic mice (Mercer et al., 1991; Kapur et al., 1991). In this paper, expression of the transgene, D beta H-nlacZ, in the gastrointestinal tract is characterized in more detail in wild-type mice and mice which are also homozygous for the lethal spotted allele (ls). Expression of the transgene in wild-type embryos was first detected in scattered mesenchymal cells in the proximal foregut on embryonic day 9.5, and progressed distally until embryonic day 13.5 when the entire length of the gut was colonized by such cells. Several observations suggest that the mesenchymal cells which express the transgene (MCET) are, in fact, enteric neuroblasts, probably derived from the vagal neural crest. (1) The presence of MCET in progressively more caudal portions of the embryonic gut correlated with the neurogenic potential of isolated gastrointestinal segments grafted under the renal capsule. (2) Mitotic activity of MCET was demonstrated by incorporation of [3H]thymidine in utero. (3) The migratory behavior of MCET and/or their precursors was revealed in anastomotic subcapsular grafts of gut from transgenic and non-transgenic embryos; enteric ganglia of the latter were populated by MCET from the former. (4) Enteric expression of the transgene postnatally was restricted to intrinsic neurons that coexpressed other phenotypic markers of neuronal differentiation. The pattern of transgene expression in ls/ls mice was identical to that seen in ls/+ and +/+ mice until embryonic day 12.5.(ABSTRACT TRUNCATED AT 250 WORDS)

During early development, there are numerous instances where a bipotent progenitor divides to give rise to two progeny cells with different fates. The Notch gene of Drosophila and its homologues in other metazoans have been implicated in many of these cell fate decisions. It has been argued that the role of Notch in such instances may be to maintain cells in a precursor state susceptible to specific differentiating signals. This has been difficult to prove, however, due to a lack of definitive markers for precursor identity. We here perform molecular and morphological analyses of the roles of Notch in ovarian follicle cells during Drosophila oogenesis. These studies show directly that constitutively active Notch arrests cells at a precursor stage, while the loss of Notch function eliminates this stage. Expression of moderate levels of activated Notch leads to partial transformation of cell fates, as found in other systems, and we show that this milder phenotype correlates with a prolonged, but still transient, precursor stage. We also find that expression of constitutively active Notch in follicle cells at later stages leads to a defect in the anterior-posterior axis of the oocyte.

1. The potency of agonists and competitive antagonists on the two expressed forms of the nicotinic acetylcholine receptor (adult or junctional subtype, ε-AChR; fetal or extrajunctional subtype, γ-AChR) have not previously been compared systematically in homogeneous receptor preparations. 2. Each subtype of the receptor was expressed separately in Xenopusoocytes by cytoplasmic injection of combinations of RNA transcribed in vitro. The presence of each type of receptor was confirmed by single-channel recordings. Expressing oocytes were assayed using discontinuous, single-electrode voltage clamp by measuring peak currents in response to test compounds. 3. The extrajunctional subtype was more potently activated by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP) than was the junctional form. There was no statistically significant difference in potency between the two subtypes for other nicotinic agonists (nicotine, cytisine and succinylcholine). The rank order of potency for ε-AChR was succinylcholine>cytisine>DMPP>nicotine, and that for γ-AChR was DMPP>cytisine>succinylcholine>nicotine. 4. Two agonists (cytisine and succinylcholine) displayed six- to eight-fold greater intrinsic activity in activating ε-AChR over γ-AChR. There was no difference between the two forms of receptor in efficacy for nicotine. 5. The extrajunctional form was much more potently inhibited by the steroidal competitive antagonist pancuronium than was the junctional receptor. However, there was no significant difference in potency of inhibition by the curariform drug atracurium. 6. Contrary to previous reports, there is no consistent relation between the effect of agonists and antagonists and the subtype of receptor. These data suggest that the resistance or sensitivity to these agents seen in various clinical settings are related to other cellular factors.

A hybrid gene containing 182 codons of Escherichia coli beta-lactamase at the amino terminus of the corresponding protein and 141 codons of alpha-globin at the carboxyl terminus was generated by inserting chimpanzee alpha-globin cDNA into the Pst I site of plasmid pBR322. RNA transcribed in vitro from this plasmid gave a corresponding hybrid protein in a wheat germ cell-free translation system. The hybrid protein was protected from tryptic digestion and the pre-beta-lactamase signal peptide was removed when dog pancreas membrane vesicles were present during translation. A deletion mutant containing 23 codons of pre-beta-lactamase signal sequence and 5 codons of mature beta-lactamase fused to the alpha-globin cDNA gave a shorter hybrid protein that behaved similarly. However, a mutation that removed essentially all of the pre-beta-lactamase sequence gave a protein that was neither protected nor processed. Hence, at most, only the signal peptide and the first 5 amino acids of beta-lactamase were necessary to convert alpha-globin (a cytoplasmic protein) into a secretory protein.