The death of a neonatal infant following cardiac surgery and the transfusion of packed red cells (RBCs) with high plasma potassium levels is reported. The patient had been diagnosed at 2 weeks of age as having multiple cardiac malformations. During cardiopulmonary bypass surgery, multiple units of packed RBCs less than 5 days old were transfused. In response to a "stat" order and after depletion of stock units prepared for neonatal usage, a 32-day-old unit of packed RBCs was issued for transfusion. After approximately 60 mL was rapidly transfused from this unit, the patient experienced cardiac arrest. Serum potassium concentration after transfusion and before death was 8.9 mmol per L. Plasma potassium concentration in the remainder of the transfused packed RBC unit was approximately 60 mmol per L. A model was created to calculate the posttransfusion plasma potassium concentration, and close correlation was found between the model and the observed potassium concentration, which assumes that the potassium load had not yet been distributed to the extravascular and intracellular fluid compartments. It is concluded that the transfusion of relatively large volumes of RBCs be limited to fresh packed RBCs or to packed RBCs that have been saline washed, to minimize the complications of electrolyte disturbances.

24Asthma is an inflammatory disease of the lung characterized by airways 25 hyperresponsiveness, inflammation, and mucus hyperproduction. Current mainstream 26 therapies include bronchodilators that relieve bronchoconstriction, and inhaled 27 glucocorticoids to reduce inflammation. The small molecule hormone and 28 neurotransmitter serotonin has long been known to be involved in inflammatory 29 processes; however, its precise role in asthma is unknown. We have previously 30 established that activation of serotonin 5-HT 2A receptors has potent anti-inflammatory 31 activity in primary cultures of vascular tissues, and in the whole animal in vasculature 32 and gut tissues. The 5-HT 2A receptor agonist, (R)-2,5-Dimethoxy-4-iodoamphetamine 33 ((R)-DOI) is especially potent. In this work, we have examined the effect of (R)-DOI in 34 an established mouse model of allergic asthma. In the ovalbumin mouse model of 35allergic inflammation, we demonstrate that inhalation of (R)-DOI prevents the 36 development of many key features of allergic asthma including airways 37 hyperresponsiveness, mucus hyperproduction, airways inflammation, and pulmonary 38 eosinophil recruitment. Our results highlight a likely role of the 5-HT 2 receptors in 39 allergic airways disease, and suggest that 5-HT 2 receptor agonists may represent an 40 effective and novel small-molecule based therapy for asthma. 41 42

Muere C, Neumueller S, Miller J, Olesiak S, Hodges MR, Pan L, Forster HV. Atropine microdialysis within or near the pre-Bötzinger Complex increases breathing frequency more during wakefulness than during NREM sleep. J Appl Physiol 114: 694-704, 2013. First published December 27, 2012 doi:10.1152/japplphysiol.00634.2012.-Normal activity of neurons within the medullary ventral respiratory column (VRC) in or near the pre-Bötzinger Complex (preBötC) is dependent on the balance of inhibitory and excitatory neuromodulators acting at their respective receptors. The role of cholinergic neuromodulation during awake and sleep states is unknown. Accordingly, our objective herein was to test the hypotheses that attenuation of cholinergic modulation of VRC/ preBötC neurons in vivo with atropine would: 1) decrease breathing frequency more while awake than during non-rapid-eye-movement (NREM) sleep and 2) increase other excitatory neuromodulators. To test these hypotheses, we unilaterally dialyzed mock cerebrospinal fluid (mCSF) or 50 mM atropine in mCSF in or near the preBötC region of adult goats during the awake (n ϭ 9) and NREM sleep (n ϭ 7) states. Breathing was monitored, and effluent dialysate was collected for analysis of multiple neurochemicals. Compared with dialysis of mCSF alone, atropine increased (P Ͻ 0.05) breathing frequency while awake during the day [ϩ10 breaths (br)/min] and at night (ϩ9 br/min) and, to a lesser extent, during NREM sleep (ϩ5 br/min). Atropine increased (P Ͻ 0.05) effluent concentrations of serotonin (5-HT), substance P (SP), and glycine during the day and at night. When atropine was dialyzed in one preBötC and mCSF in the contralateral preBötC, 5-HT and SP increased only at the site of atropine dialysis. We conclude: 1) attenuation of a single neuromodulator results in local changes in other neuromodulators that affect ventilatory control, 2) effects of perturbations of cholinergic neuromodulation on breathing are state-dependent, and 3) interpretation of perturbations in vivo requires consideration of direct and indirect effects.neuromodulators; control of breathing; acetylcholine; non-rapid-eyemovement sleep THE NEUROMODULATOR ACETYLCHOLINE (ACh) is involved in a variety of central and peripheral neural circuits, including the control of breathing and sleep (7,12,16,19,25,39). The effects of ACh are mediated through either nicotinic or muscarinic receptors coupled to second messenger pathways (6, 10, 13, 42). There are five known muscarinic ACh receptor (mAChR) subtypes (M 1 to M 5 ) expressed in the brain stem, differing in expression profiles and in effects on breathing (2,9,11,13,33,34). For example, the pontine respiratory group (PRG) nuclei express M 1 -M 3 mAChRs (34). The predominant effect of cholinergic modulation of PRG neurons within the Kölliker-Fuse Nucleus (KFN) appears to be excitatory, since reverse dialysis of the nonselective mAChR antagonist atropine reduced minute ventilation (V I ) and breathing frequency in both the awake state and during non-rapid-eye-movement (NREM...

Background The efficacy of opioid administration to reduce postoperative pain is limited by respiratory depression. We investigated whether clinically relevant opioid concentrations altered the respiratory pattern in the parabrachial nucleus, a pontine region contributing to respiratory pattern generation, and compared these effects with a medullary respiratory site, the pre-Bötzinger complex. Methods Studies were performed in 40 young and 55 adult artificially ventilated, decerebrate rabbits. We identified an area in the parabrachial nucleus where α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid microinjections elicited tachypnea. Two protocols were performed in separate sets of animals. First, bilateral microinjections of the μ-opioid receptor agonist [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin (100 μM) into the “tachypneic area” determined the effect of maximal μ-opioid receptor activation. Second, respiratory rate was decreased with continuous IV infusions of remifentanil. The opioid antagonist naloxone (1 mM) was then microinjected bilaterally into the “tachypneic area” of the parabrachial nucleus to determine whether the respiratory rate depression could be locally reversed. Results Average respiratory rate was 27 ± 10 breaths/min. First, [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin injections decreased respiratory rate by 62 ± 20% in young and 45 ± 26% in adult rabbits (both P < 0.001). Second, during IV remifentanil infusion, bilateral naloxone injections into the “tachypneic area” of the parabrachial nucleus reversed respiratory rate depression from 55 ± 9% to 20 ± 14% in young and from 46 ± 20% to 18 ± 27% in adult rabbits (both P < 0.001). The effects of bilateral [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin injection and IV remifentanil on respiratory phase duration in the “tachypneic area” of the parabrachial nucleus was significantly different from the pre-Bötzinger complex. Conclusions The “tachypneic area” of the parabrachial nucleus is highly sensitive to μ-opioid receptor activation and mediates part of the respiratory rate depression by clinically relevant administration of opioids.

Background The preBötzinger Complex (preBC) plays an important role in respiratory rhythm generation. This study was designed to determine whether the preBC mediated opioid-induced respiratory rate depression at clinically relevant opioid concentrations in vivo and whether this role was age-dependent. Methods Studies were performed in 22 young and 32 adult New Zealand White rabbits. Animals were anesthetized, mechanically ventilated and decerebrated. The preBC was identified by the tachypneic response to injection of D,L-homoysteic acid. (1) The mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO, 100μM) was microinjected into the bilateral preBC and reversed with naloxone (1mM) injection into the preBC. (2) Respiratory depression was achieved with intravenous remifentanil (0.08–0.5 mcg/kg/min). Naloxone (1mM) was microinjected into the preBC in an attempt to reverse the respiratory depression. Results (1) DAMGO injection depressed respiratory rate by 6 ± 8 breaths/min in young and adult rabbits (mean ± SD, p <0.001). DAMGO shortened the inspiratory and lengthened the expiratory fraction of the respiratory cycle by 0.24 ± 0.2 in adult and young animals (p <0.001). (2) During intravenous remifentanil infusion, local injection of naloxone into the preBC partially reversed the decrease in inspiratory fraction/increase in expiratory fraction in young and adult animals (0.14 ± 0.14, p <0.001), but not the depression of respiratory rate (p = 0.19). PreBC injections did not affect respiratory drive. In adult rabbits, the contribution of non-preBC inputs to expiratory phase duration was larger than preBC inputs (3.5(−5.2–1.1), median (25%–75%), p = 0.04). Conclusions Systemic opioid effects on respiratory phase timing can be partially reversed in the preBC without reversing the depression of respiratory rate.

The survival rate for patients with oral squamous cell carcinoma (OSCC) has not seen marked improvement in recent decades despite enhanced efforts in prevention and the introduction of novel therapies. We have reported that pharmacological exacerbation of the unfolded protein response (UPR) is an effective approach to killing OSCC cells. The UPR is executed via distinct signaling cascades whereby an initial attempt to restore folding homeostasis in the endoplasmic reticulum during stress is complemented by an apoptotic response if the defect cannot be resolved. To identify novel small molecules able to overwhelm the adaptive capacity of the UPR in OSCC cells, we engineered a complementary cell-based assay to screen a broad spectrum of chemical matter. Stably transfected CHO-K1 cells that individually report (luciferase) on the PERK/eIF2α/ATF4/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR pathways, were engineered [1]. The triterpenoids dihydrocelastrol and celastrol were identified as potent inducers of UPR signaling and cell death in a primary screen and confirmed in a panel of OSCC cells and other cancer cell lines. Biochemical and genetic assays using OSCC cells and modified murine embryonic fibroblasts demonstrated that intact PERK-eIF2–ATF4-CHOP signaling is required for pro-apoptotic UPR and OSCC death following celastrol treatment.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

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