The effect of the natural bile acid, taurolithocholic acid 3‐sulfate (TLC‐S), on calcium signalling in pancreatic acinar cells has been investigated. TLC‐S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC‐S in a calcium‐free external solution. TLC‐S‐induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC‐S application, displayed vectorial apical‐to‐basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC‐S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid‐induced cellular damage.

Here we show that endothelial nitric oxide synthase (eNOS) is a critical regulator of this pathway. ICAM-1 stimulated eNOS by a mechanism that was clearly distinct from that utilized by insulin. In particular, phosphorylation of eNOS on S1177 in response to ICAM-1 activation was regulated by src family protein kinase, rho GTPase, Ca 2؉ , CaMKK, and AMPK, but not Akt/PI3K. Functional neutralization of any component of this pathway or its downstream effector guanylyl cyclase significantly reduced lymphocyte diapedesis across the endothelial monolayer. In turn, activation of NO signaling promoted lymphocyte transmigration. The eNOS signaling pathway was required for T-cell transmigration across primary rat and human microvascular endothelial cells and also when shear flow was applied, suggesting that this pathway is ubiquitously used. These data reveal a novel and essential role of eNOS in basic immune function and provide a key link in the molecular network governing endothelial cell compliance to diapedesis. INTRODUCTIONThe events controlling the capture and subsequent migration of circulating lymphocytes across the vascular wall have been studied extensively, and many of its generic principles are known. However, the signaling mechanisms that underpin this process remain poorly defined. Endothelial cells (ECs) actively participate in directing and regulating the process of lymphocyte migration across the vascular wall via adhesion molecules such as vascular cell adhesion cell molecule 1 (VCAM-1; Engelhardt, 2006), platelet and endothelial cell adhesion molecule-1 (PECAM-1; Liao et al., 1997), and intercellullar adhesion molecule 1 (ICAM-1; Turowski et al., 2005). In the CNS, where ECs form a tight specialized barrier, lymphocyte recruitment utilizes the same adhesion molecules, with the pairing of lymphocyte function-associated antigen 1 (LFA-1)/ICAM-1 being preeminent in controlling diapedesis (Greenwood et al., 1995;Turowski et al., 2005).Previous studies have also shown that ICAM-1 is not only involved in the adhesion process, but its engagement activates diverse signaling pathways in ECs, some of which are required for subsequent migration. For example, ICAM-1 has been reported to interact with members of the ezrin/ radixin/moesin (ERM) family, to activate rho GTPases, src, protein kinase C (PKC), and mitogen-activated protein (MAP) kinases and a series of proteins involved in the formation of focal adhesions such as FAK, paxillin, and p130 cas (Turowski et al., 2005). A rise of intracellular calcium appears to be central to ICAM-mediated events (EtienneManneville et al., 2000) and precedes the activation of the other master regulators such as rho GTPases and PKC. Importantly, EC signaling via Ca 2ϩ and rho is essential for subsequent lymphocyte migration (Adamson et al., 1999;Etienne-Manneville et al., 2000) at least in part by regulating adherens junction modulation during paracellular diapedesis (Allingham et al., 2007;Turowski et al., 2008). Indeed, ICAM-1 engagement induces phosphorylation of vascular ...

The retinal pigment epithelium (RPE) plays a critical role in the maintenance of the outer retina. RPE cell death or dysfunction drives the pathophysiology of many retinal diseases, but the physiological response of the retina to RPE cell loss is poorly understood, mainly because of the absence of suitable experimental models. Here, we generated a transgenic mouse in which an inducible Cre recombinase is expressed exclusively in the RPE under the control of the monocarboxylate transporter 3 gene promoter (RPE CreER ). This was crossed with a transgenic mouse harboring a diphtheria toxin A (DTA) chain gene rendered transcriptionally silent by a floxed stop sequence. We show that activation of DTA in the double transgenic mouse (RPE CreER /DTA) led to 60 -80% RPE cell death, with surviving cells maintaining the integrity of the monolayer by increasing their size. Despite the apparent morphological normality of the enlarged RPE cells in the RPE CreER /DTA mice, functional analysis revealed significant deficits on electroretinography, and retinal histopathology showed regions of photoreceptor rosetting and degeneration although with retention of a normal vascular network. Our study reveals that whilst the RPE monolayer has a remarkable intrinsic capacity to cope with cellular attrition, specific aspects of RPE multifunctionality essential for photoreceptor survival are compromised. The RPE CreER /DTA mouse offers advantages over models that employ chemical or mechanical strategies to kill RPE cells, and should be useful for the development and evaluation of RPE-based therapies, such as stem cell transplantation.cell death ͉ pigment ͉ retina

During the early development of the nervous system, ␥-aminobutyric acid (GABA) type A receptor (GABA A R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA A R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca 2؉ influx through L-type voltage-gated Ca 2؉channels. We further demonstrate that GABA A R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA A Rs. This BDNF/TrkB-dependent increase in surface levels of GABA A Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA A R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA A R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

Purpose In the critically ill, hospital-acquired bloodstream infections (HA-BSI) are associated with significant mortality. Granular data are required for optimizing management, and developing guidelines and clinical trials. Methods We carried out a prospective international cohort study of adult patients (≥ 18 years of age) with HA-BSI treated in intensive care units (ICUs) between June 2019 and February 2021. Results 2600 patients from 333 ICUs in 52 countries were included. 78% HA-BSI were ICU-acquired. Median Sequential Organ Failure Assessment (SOFA) score was 8 [IQR 5; 11] at HA-BSI diagnosis. Most frequent sources of infection included pneumonia (26.7%) and intravascular catheters (26.4%). Most frequent pathogens were Gram-negative bacteria (59.0%), predominantly Klebsiella spp. (27.9%), Acinetobacter spp . (20.3%), Escherichia coli (15.8%), and Pseudomonas spp . (14.3%). Carbapenem resistance was present in 37.8%, 84.6%, 7.4%, and 33.2%, respectively. Difficult-to-treat resistance (DTR) was present in 23.5% and pan-drug resistance in 1.5%. Antimicrobial therapy was deemed adequate within 24 h for 51.5%. Antimicrobial resistance was associated with longer delays to adequate antimicrobial therapy. Source control was needed in 52.5% but not achieved in 18.2%. Mortality was 37.1%, and only 16.1% had been discharged alive from hospital by day-28. Conclusions HA-BSI was frequently caused by Gram-negative, carbapenem-resistant and DTR pathogens. Antimicrobial resistance led to delays in adequate antimicrobial therapy. Mortality was high, and at day-28 only a minority of the patients were discharged alive from the hospital. Prevention of antimicrobial resistance and focusing on adequate antimicrobial therapy and source control are important to optimize patient management and outcomes. Supplementary Information The online version contains supplementary material available at 10.1007/s00134-022-06944-2.