Neutrophils are crucial mediators of host defense that are recruited to the central nervous system (CNS) in large numbers during acute bacterial meningitis caused by Streptococcus pneumoniae . Neutrophils release neutrophil extracellular traps (NETs) during infections to trap and kill bacteria. Intact NETs are fibrous structures composed of decondensed DNA and neutrophil-derived antimicrobial proteins. Here we show NETs in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis, and their absence in other forms of meningitis with neutrophil influx into the CSF caused by viruses, Borrelia and subarachnoid hemorrhage. In a rat model of meningitis, a clinical strain of pneumococci induced NET formation in the CSF. Disrupting NETs using DNase I significantly reduces bacterial load, demonstrating that NETs contribute to pneumococcal meningitis pathogenesis in vivo. We conclude that NETs in the CNS reduce bacterial clearance and degrading NETs using DNase I may have significant therapeutic implications.

Swärd. Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol 291: C1326 -C1335, 2006; doi:10.1152/ajpcell.00046.2006.-Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca 2ϩ sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were ϳ15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTP␥S was increased in KO compared with WT as shown using a pull-down assay. Following ␣-toxin permeabilization, no difference in Ca 2ϩ sensitivity or in Ca 2ϩ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased ␣1-adrenergic contraction. Following inhibition of PKC, ␣1-adrenergic contraction was normalized. PDBu-induced Ca 2ϩ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca 2ϩ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca 2ϩ sensitivity. Ca 2ϩ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor CAVEOLAE are 50 -100 nm flask-shaped membrane invaginations that are abundant in endothelial cells, adipocytes, and smooth muscle cells. Caveolae are characterized by high cholesterol and sphingolipid content, and a light buoyant density. They are stabilized by the caveolin proteins (9). Specific G protein-coupled and tyrosine kinase receptors, as well as downstream signaling intermediaries, have been shown to be caveolae associated (26,30). Such clustering has been envisioned to facilitate receptor signaling and has been proposed to play a role in receptor internalization. The scaffolding domain of caveolin-1 (Cav-1) may also function as a broad-spectrum kinase inhibitor (9).Compared with endothelial and adipocyte caveolae, smooth muscle caveolae have received relatively little attention. With the use of cholesterol depletion to disrupt caveolae in denuded caudal arteries from the rat, we demonstrated that serotonin (5-HT 2A ) as well as endothelin-1 (ET-1) receptor signaling was impaired by cholesterol depletion. Moreover, restoration of caveolae by cholest...

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMPprotein kinase A) seems to exist. We have explored cGMP-and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI؊/؊). In intact ileum strips of wild type mice (cGKI؉/؉), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by ϳ80%. The initial peak was less inhibited (ϳ30%). This relaxation was associated with a reduction in intracellular [Ca 2؉ ] and decreased Ca 2؉ sensitivity. Contractions of cGKI؊/؊ ileum were not influenced by 8-Br-cGMP. EC 50 for 8-Br-cGMP for PKG was estimated to be 10 nM. PKGindependent relaxation by 8-Br-cGMP had an EC 50 of 10 M. Relaxation by cAMP (ϳ50% at 100 M), Ca 2؉ sensitivity of force, and force potentiation by GTP␥S were similar in cGKI؉/؉ and cGKI؊/؊ tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca 2؉ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca 2؉ sensitivity.The cGMP-dependent protein kinase (PKG) 1 is the main mediator of nitric oxide (NO) induced relaxation of smooth muscle (1, 2). Although this pathway is well established, the cellular actions of PKG, NO, and cGMP are complex and not fully understood. In addition to the effects via PKG, both NO and cGMP can have direct effects on other systems in the cell, e.g. NO on cADP-ribose signaling or on Ca 2ϩ handling (3, 4) or cGMP on ion channels. A cross-talk between the cGMP and cAMP pathways has been proposed to exist. This can involve cross-activation of PKG by cAMP and of cAMP-dependent protein kinase (PKA) by cGMP as well as actions of the nucleotides or the kinases on phosphodiesterases (21-24).Recently, a mouse strain lacking cGKI, which is the PKG isoform normally present in smooth muscle, was generated (7). Intact smooth muscles of these animals are not relaxed by NO or cGMP, but their responses to cAMP are essentially unaltered (7,8,25). However, a detailed characterization of the cGMP and cAMP dependencies is difficult in intact tissue in which diffusion and metabolism of nucleotide analogues might influence the responses, and the effects on Ca 2ϩ sensitivity cannot be directly assessed and distinguished from effects on intracellular Ca 2ϩ levels. We have explored cGMP-and cAMP-dependent relaxation mechanisms of smooth muscle using PKG-deficient (cGKIϪ/Ϫ) and wild type (cGKIϩ/ϩ) mice. The effects of cGMP on intracellular [Ca 2ϩ ] and force were examined in intact preparations of small intestine. Using ␣-toxin-permeabilized preparations in which the concentration...

Ca2؉ sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca 2؉ sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving ϳ50% of maximal active force were compared in small intestinal preparations: 1) Ca 2؉ -activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2) Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5-3-O-(thio)triphosphate); and 3) PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca 2؉ sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca 2؉ sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca 2؉ sensitization in smooth muscle.Smooth muscle contraction involves a rise in intracellular [Ca 2ϩ ]. The Ca 2ϩ -calmodulin complex activates myosin light chain kinase, which phosphorylates the 20-kDa myosin regulatory light chains (MLC 20 MLCP (PP1M) belongs to the PP1 group of phosphatases and is a heteromeric enzyme consisting of three subunits: a catalytic subunit (PP1M C ); a myosin-targeting subunit (MYPT1); and the M20 subunit (5). MLCP has been reported to be inhibited directly by phosphorylation of its myosin-targeting subunit, MYPT1, on residue Thr-695 performed by Rho kinase (6), MYPT1-associated kinase (sometimes referred to as ZIP-like kinase) (7), and integrin-linked kinase (8). Phosphorylation of MYPT1 at Thr-34 by protein kinase C (PKC) has been shown in vitro (9); the relevance of this result and the effect on the holoenzyme are unknown. MLCP is blocked and inhibited by the binding of the phosphorylated form of protein kinase Cpotentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17). CPI-17 is a major target for phosphorylation by PKC, which phosphorylates the Thr-38 residue. This process has been shown to be crucial for PKCmediated increase in Ca 2ϩ sensitivity of force (Ca 2ϩ sensitization) in smooth muscle (10,36,43). In vitro, Thr-38 on CPI-17 is also phosphorylated by Rho kinase (11), MYPT1-associated kinase (12), protein kinase N (13), integrin-linked kinase (14), p21-activated protein kinase (15), cAMP-activated protein kinase A (16), and cGMP-activated pro...

Background Optimal infusion rate of colloids in patients with suspected hypovolemia is unknown, and the primary objective of the present study was to test if plasma volume expansion by 5% albumin is greater if fluid is administered slowly rather than rapidly. Methods Patients with signs of hypoperfusion after major abdominal surgery were randomized to intravenous infusion of 5% albumin at a dose of 10 ml/kg (ideal body weight) either rapidly (30 min) or slowly (180 min). Plasma volume was measured using radiolabeled albumin at baseline, at 30 min, and at 180 min after the start of infusion. Primary outcome was change in plasma volume from the start of infusion to 180 min after the start of infusion. Secondary outcomes included the change in the area under the plasma volume curve and transcapillary escape rate (TER) for albumin from 180 to 240 min after the start of albumin infusion. Results A total of 33 and 31 patients were included in the analysis in the slow and rapid groups, respectively. The change in plasma volume from the start of infusion to 180 min did not differ between the slow and rapid infusion groups (7.4 ± 2.6 vs. 6.5 ± 4.1 ml/kg; absolute difference, 0.9 ml/kg [95%CI, − 0.8 to 2.6], P = 0.301). Change in the area under the plasma volume curve was smaller in the slow than in the rapid infusion group and was 866 ± 341 and 1226 ± 419 min ml/kg, respectively, P < 0.001. TER for albumin did not differ and was 5.3 ± 3.1%/h and 5.4 ± 3%/h in the slow and in the rapid infusion groups, respectively, P = 0.931. Conclusions This study does not support our hypothesis that a slow infusion of colloid results in a greater plasma volume expansion than a rapid infusion. Instead, our result of a smaller change in the area under the plasma volume curve indicates that a slow infusion results in a less efficient plasma volume expansion, but further studies are required to confirm this finding. A rapid infusion has no effect on vascular leak as measured after completion of the infusion. Trial registration EudraCT2013-004446-42 registered December 23, 2014. Electronic supplementary material The online version of this article (10.1186/s13054-019-2477-7) contains supplementary material, which is available to authorized users.