Cellular immunophenotype of major spine surgery in adults.

PURPOSE: ASD reconstructions are a major, sterile traumatic insult, likely causing perturbations to the immune systems. The immune response to surgery is associated with outcomes. The purpose of this study was to examine for a detectable immune signature associated with ASD surgery. METHODS: Consecutive patients undergoing ASD surgery were approached and enrolled. Peripheral blood was drawn before incision, 4 h after, and 24 h after incision. Blood was stabilized and comprehensive flow cytometric immunophenotyping performed. Leukocyte population frequency, absolute number and activation marker expression were defined. Immunologic features were defined and analyzed by hierarchical clustering and principal component analysis (PCA). Changes over time were evaluated by repeated measures ANOVA (RMANOVA) and were corrected for a 1% false discovery rate. Post hoc testing was by Dunn's test. p values of < = 0.05 were considered significant. RESULTS: Thirteen patients were enrolled; 11(85%) F, 65.4 years (± 7.5), surgical duration 418 ± 83 min, EBL 1928 ± 1253 mL. Hierarchical clustering and PCA found consistent time from incision-dependent changes. HLA-DR and activating co-stimulatory molecule CD86 were depressed at 4 h and furthermore at 24 h on monocyte surfaces. CD4 + HLA-DR + T cells, but not CD8 +, increased over time with increased expression of PD-1 at 4 and 24 h. CONCLUSIONS: Despite surgery and patient heterogeneity, we identified an immune signature associated with the sterile trauma of ASD surgery. Circulating leukocyte populations change in composition and signaling protein expression after incision and persisting to 24 h after incision, suggesting an immunocompromised state. Further work may determine relationships between this state and poor outcomes after surgery.

Dysregulation of the leukocyte signaling landscape during acute COVID-19.

The global COVID-19 pandemic has claimed the lives of more than 750,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. We find that although COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations, STAT3 phosphorylation predominated in both monocytes and T cells. STAT3 phosphorylation was tightly correlated with circulating IL-6 levels and high levels of phospho-STAT3 was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-γ production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.

Dysregulation of the Leukocyte Signaling Landscape during Acute COVID-19.

The global COVID-19 pandemic has claimed the lives of more than 450,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated local tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations. STAT3 phosphorylation predominated in both monocytes and T cells and was tightly correlated with circulating IL-6 levels. High levels of STAT3 phosphorylation was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-gamma production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.

The effect of bladder outlet obstruction on alpha1- and beta-adrenoceptor expression and function.

AIMS: To explore possible changes in expression and/or function of alpha(1)- and beta-adrenoceptor subtypes as a cause for bladder dysfunction in a rat model of bladder outlet obstruction (BOO). METHODS: BOO was induced in rats by partial urethral ligature. Contraction and relaxation experiments were performed with isolated bladder strips from BOO, sham-operated and non-operated (control) rats 7 days after BOO induction. mRNA expression of alpha(1)- and beta-adrenoceptor subtypes was assessed by quantitative real-time PCR. RESULTS: Receptor-independent contraction or relaxation did not differ between BOO and sham rats. The alpha(1)-agonists methoxamine and A-61,603 caused only weak contraction without major differences between groups. Against KCl-induced tone, the beta-adrenoceptor agonists noradrenaline and isoprenaline caused similar relaxation in BOO and sham rats, whereas relaxation in response to the beta(3)-selective BRL 37,344 was attenuated. Against passive tension, noradrenaline induced relaxation in sham and control rats; in contrast, noradrenaline induced contraction at low concentrations and relaxation at high concentrations in BOO rats. The contraction component was abolished by the alpha(1)-antagonist prazosin. The mRNA expression of alpha(1D)-adrenoceptors was increased in BOO, whereas none of the other receptor mRNAs were up-regulated. CONCLUSIONS: In a rat BOO model, weak contraction responses to alpha(1)-agonists and relaxation responses to beta-agonists are not altered to a major extent. Nevertheless, relaxation responses to the endogenous agonist noradrenaline are turned into alpha(1)-adrenoceptor-mediated contraction responses in BOO, possibly due to an up-regulation of alpha(1D)-adrenoceptors.

Signal transduction underlying the control of urinary bladder smooth muscle tone by muscarinic receptors and beta-adrenoceptors.

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M3 subtype, with the M2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by beta-adrenoceptors, in most species involving a strong beta3-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of beta-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to beta-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in beta-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and beta-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder.

Effects of gender, age and hypertension on beta-adrenergic receptor function in rat urinary bladder.

beta-Adrenoceptors mediate urinary bladder relaxation, and gender, age and hypertension have been linked to bladder dysfunction. Therefore, we have studied whether any of these factors affects the ability of beta-adrenoceptor agonists to relax rat bladder detrusor muscle in vitro. For this purpose we have compared male and female Wistar rats, young and old male Wistar rats, and male normotensive and spontaneously hypertensive rats (SHR). Comparisons were done using KCl-precontracted bladder strips (length about 15-20 mm) and the endogenous agonist noradrenaline, the synthetic non-subtype-selective agonist isoprenaline, and the prototypical beta(3)-adrenoceptor agonists BRL 37,344 and CGP 12,177. While all agonists yielded numerically weaker relaxation in female as compared to male rats (for example for noradrenaline E(max) 40+/-4% vs 53+/-6% relaxation, pEC(50) 5.41+/-0.13 vs 5.60+/-0.14), this difference reached statistical significance only for the weak partial agonist CGP 12,177. Responses to all agonists were attenuated in old as compared to young rats, largely due to a reduced maximum effect, although the difference did not reach statistical significance for isoprenaline. The maximum relaxation responses to noradrenaline and isoprenaline were significantly lower in SHR than in normotensive rats, but both strains exhibited similar responses to the partial agonist BRL 37,344. We conclude that factors associated with bladder dysfunction, such as gender, age and hypertension, can be associated with impaired beta-adrenoceptor-mediated bladder relaxation. However, these alterations are not always consistent across various agonists, and the extent of the differences can be small. Therefore, we propose that beta-adrenoceptor dysfunction may contribute to the pathophysiology of such conditions, but is unlikely to be the only or even the major factor in this regard. We speculate that beta-adrenoceptor agonists may be effective in the treatment of bladder dysfunction under all of these conditions.

Lack of evidence that nebivolol is a ß3-adrenoceptor agonist.

Nebivolol is a selective ß1-adrenoceptor antagonist which, in addition, displays endothelium-dependent vasodilating properties in humans and other species. ß3-adrenoceptors have been proposed to be a molecular target of nebivolol-induced vasodilatation. Therefore, we have investigated possible ß3-adrenoceptor agonism by nebivolol for relaxation of the human and rat urinary bladder (prototypical ß3-adrenoceptor-mediated responses) as well as for cAMP accumulation in Chinese hamster ovary cells stably transfected with the human ß-adrenoceptor subtypes. Nebivolol concentration-dependently relaxed both human and rat isolated urinary bladder strips but with low potency, similar to that reported for vasodilatation. However, nebivolol-induced bladder relaxation in either species was not inhibited by the ß3-adrenoceptor antagonist SR 59,230A (10µM), although this compound inhibited the isoprenaline-induced relaxation with the expected potency. In radioligand binding studies nebivolol had lower affinity for human ß3-adrenoceptors than the other two ß-adrenoceptor subtypes, but this low affinity was in line with its potency to relax the bladder or isolated blood vessels. In functional studies nebivolol even in high concentrations did not stimulate cAMP formation via any of the three cloned human ß-adrenoceptors or in rat bladder smooth muscle cells. Taken together these data demonstrate that nebivolol can relax not only vascular but also urinary bladder smooth muscle. However, they do not support the hypothesis that nebivolol is an agonist at cloned human ß3-adrenoceptors or in rat or human urinary bladder.

Age-dependent regulation of skeletal muscle mitochondria by the thrombospondin-1 receptor CD47.

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3 month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O(2) ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12 month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1-CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

WITHDRAWN: Is nebivolol a beta(3)-adrenoceptor agonist?

This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Does phospholipase C mediate muscarinic receptor-induced rat urinary bladder contraction?

Muscarinic acetylcholine receptors, particularly M(3) receptors, are physiologically the most important mechanism to induced urinary bladder smooth muscle contraction. Their prototypical signaling response is a stimulation of phospholipase C (PLC), and this also has been shown in the urinary bladder. Nevertheless, it has remained controversial whether PLC signaling mediates bladder contraction induced by muscarinic receptor agonists. Studies in favor and against a role for PLC differed in their experimental protocol (single versus repeated concentration-response curves within a single preparation) and in the PLC inhibitors that have been used. We have now tested whether previous differential conclusions regarding a role for PLC are related to inhibitors and/or experimental protocols. In a single curve protocol, U-73,122 [1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione] did not attenuate carbachol responses. In a repeated curve protocol, ET-18-OCH(3) (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine) lacked significant inhibition relative to vehicle time controls. In contrast, D609 (O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt) depressed maximal carbachol effects but also nonspecifically inhibited contraction induced by KCl. Neomycin did not affect the carbachol-induced rat urinary bladder contraction. We conclude that previously reported differences relate to the use of inhibitors rather than experimental protocols and that the overall data do not support a role for PLC in M(3) muscarinic receptor-mediated rat bladder contraction.

Does cyclic AMP mediate rat urinary bladder relaxation by isoproterenol?

Cyclic AMP is the prototypical second messenger of beta-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon beta-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying beta-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 microM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 microM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 microM), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 30 microM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 microM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 microM), tetraethylammonium (3 microM), apamin (300 nM), and glibenclamide (10 microM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in beta-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.