Low mortality rates among critically ill adults with COVID-19 at three non-academic intensive care units in south Sweden.

BACKGROUND: The COVID-19 pandemic has put an exceptional strain on intensive care units worldwide. During the first year, the survival of patients with acute hypoxaemic respiratory failure appears to have improved. We aimed to describe the mortality rates, management characteristics and two pandemic waves during the first year at three non-academic rural intensive care units in Sweden. METHODS: We retrospectively analysed all cases of COVID-19 admitted to intensive care units in Region Jönköping County during 1 year. The primary endpoint was 30-day mortality. RESULTS: Between 14th March 2020 and 13th March 2021, 264 patients were admitted to undergo intensive care with confirmed SARS-CoV-2 infection. The 30-day mortality rate after the initial intensive care admission was 12.9%, and this rate remained unchanged during both pandemic waves. However, we found several distinct differences between the two pandemic waves, including an increase in the use of high-flow nasal oxygen but a decrease in invasive mechanical ventilation use, biochemical markers of inflammation, continuous renal replacement therapy and length of stay in the intensive care unit. CONCLUSION: Our study showed that critically ill patients with COVID-19 in Sweden have a low 30-day mortality rate which compares well with results published from academic centres and national cohorts throughout Scandinavia. During the second pandemic wave, the proportion of patients receiving invasive mechanical ventilation and continuous renal replacement therapy was lower than that in the first wave. This could be the result of increased knowledge and improved therapeutic options.

Small-Incision Lenticule Extraction (SMILE) for the Correction of Myopia with Astigmatism: Outcomes of the United States Food and Drug Administration Premarket Approval Clinical Trial.

PURPOSE: To present outcomes of the United States Food and Drug Administration premarket approval clinical trial of small-incision lenticule extraction (SMILE) for the correction of myopia and astigmatism. DESIGN: Prospective, multicenter clinical trial. PARTICIPANTS: The study included 357 eyes of 357 patients treated with SMILE (50 for myopia and 307 for myopia with astigmatism). Preoperative sphere ranged between -1.00 and -10.00 diopters (D), with manifest spherical equivalent (MSE) of up to -11.50 D and refractive cylinder of up to -3.00 D. METHODS: -Participants were followed up for 12 months. Corrected distance visual acuity (CDVA) and uncorrected distance visual acuity (UDVA), stability of the manifest refraction, and vector analysis of refractive cylinder are presented for the 307 eyes treated for myopia with astigmatism. Adverse events (AEs) are presented for all 357 eyes. MAIN OUTCOME MEASURES: Corrected distance visual acuity, uncorrected distance visual acuity, manifest refraction, astigmatic outcomes, and adverse events. RESULTS: Of the 307 astigmatic eyes enrolled in the study, 304 were treated successfully. In 3 eyes, the procedure was aborted because of intraoperative suction loss. The mean MSE reduced from -5.39±2.30 D at baseline to -0.01±0.24 D at 12 months. Of all eyes, 95.3% were within 0.50 D of emmetropia at 12 months. The percentage of eyes with UDVA of 20/20 or better was 89.0%. No loss of 2 or more lines of CDVA was observed at the 12-month visit. The refractive cylinder reduced from -1.53±0.70 D at baseline to -0.18±0.31 D at 12 months. The mean correction ratio of refractive cylinder was 0.96±0.16 and a slight undercorrection was apparent for higher attempted corrections of astigmatism. Three intraoperative AEs associated with difficult lenticule removal and resultant cap tear occurred, and all resolved without sequelae at postoperative day 1. During the postoperative period, 8 AEs were recorded, but none of them had significant consequences. CONCLUSIONS: Small-incision lenticule extraction for the treatment of myopia and astigmatism was safe and effective, and the reported AEs had no significant impact on visual acuity. Slight undercorrection of refractive cylinder requires further attention.

Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3.

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca(2+)](i)), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca(2+)](i) regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca(2+) imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca(2+)](i) and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca(2+)-dependent, TRAM-34-sensitive K(Ca)3.1 channels, and Ca(2+)-dependent, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl(-) channels. A significant portion of these Cl(-) currents can be attributed to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl(-) channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca(2+)-activated Cl(-) currents. Western blots show that K(Ca)3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both K(Ca)3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slices in situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca(2+)](i) increases, which likely play similar roles in other migratory cells in the nervous system.

Bradykinin enhances invasion of malignant glioma into the brain parenchyma by inducing cells to undergo amoeboid migration.

The molecular and cellular mechanisms governing cell motility and directed migration in response to the neuropeptide bradykinin are largely unknown. Here, we demonstrate that human glioma cells whose migration is guided by bradykinin generate bleb-like protrusions. We found that activation of the B2 receptor leads to a rise in free Ca(2+) from internal stores that activates actomyosin contraction and subsequent cytoplasmic flow into protrusions forming membrane blebs. Furthermore Ca(2+) activates Ca(2+)-dependent K(+) and Cl(-) channels, which participate in bleb regulation. Treatment of gliomas with bradykinin in situ increased glioma growth by increasing the speed of cell migration at the periphery of the tumour mass. To test if bleb formation is related to bradykinin-promoted glioma invasion we blocked glioma migration with blebbistatin, a blocker of myosin kinase II, which is necessary for proper bleb retraction. Our findings suggest a pivotal role of bradykinin during glioma invasion by stimulating amoeboid migration of glioma cells.

Toll-like receptor activation reveals developmental reorganization and unmasks responder subsets of microglia.

The sentinel and immune functions of microglia require rapid and appropriate reactions to infection and damage. Their Toll-like receptors (TLRs) sense both as threats. However, whether activated microglia mount uniform responses or whether subsets conduct selective tasks is unknown. We demonstrate that murine microglia reorganize their responses to TLR activations postnatally and that this process comes with a maturation of TLR4-organized functions. Although induction of MHCI for antigen presentation remains as a pan-populational feature, synthesis of TNFα becomes restricted to a subset, even within adult central nervous system regions. Response heterogeneity is evident ex vivo, in situ, and in vivo, but is not limited to TNFα production or to TLR-triggered functions. Also, clearance activities for myelin under physiological and pathophysiological conditions, IFNγ-enforced upregulation of MHCII, or challenged inductions of other proinflammatory factors reveal dissimilar microglial contributions. Notably, response heterogeneity is also confirmed in human brain tissue. Our findings suggest that microglia divide by constitutive and inducible capacities. Privileged production of inflammatory mediators assigns a master control to subsets. Sequestration of clearance of endogenous material versus antigen presentation in exclusive compartments can separate potentially interfering functions. Finally, subsets rather than a uniform population of microglia may assemble the reactive phenotypes in responses during infection, injury, and rebuilding, warranting consideration in experimental manipulation and therapeutic strategies.

Patient-Controlled Sedation in Port Implantation (PACSPI 1) - A feasibility trial.

Background: Central venous access is essential for the administration of chemotherapy and frequent blood sampling in patients with cancer. The subcutaneous venous port is commonly used for this purpose. Subcutaneous venous port implantation is a minor surgical procedure; however, it can provoke pain and anxiety in these vulnerable patients. The aim of this study was, before a full-scale RCT, to determine the feasibility of patient-controlled sedation with propofol and alfentanil as an adjunct to local anaesthesia during SVP implantation. Methods: We prospectively studied 40 patients scheduled for SVP implantation between 14 April 2021 and 15 October 2021 at a 500-bed secondary level hospital in Sweden. Anaesthesiologists performed subcutaneous venous port implantation with patient-controlled sedation using propofol and alfentanil. We determined pain perception (primary outcome), patient satisfaction, sedation score, and key safety measures. Results: Of the 40 patients with cancer, 80% reported a pain score ≤3 on an 11-point numeric rating scale during subcutaneous venous port implantation. Overall satisfaction with pain management and operating conditions was graded as 10 of 10 on the numeric rating scale. Four patients (10%) had bradypnoea (<8 bpm) without oxygen desaturation to ≤90%. Rescue sedation was administered to one patient (2.5%). Conclusion: Patient-controlled sedation with propofol and alfentanil during subcutaneous venous port implantation is feasible and well accepted. Ultimately the efficacy of patient-controlled sedation with propofol and alfentanil needs to be evaluated in an RCT to provide clinicians with evidence-based guidance for choosing the optimal perioperative strategy for subcutaneous venous port implantation. Clinical trial registration: NCT04631393.

Functional importance of inositol-1,4,5-triphosphate-induced intracellular Ca2+ mobilization in galanin-induced microglial migration.

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.

Differential VASP phosphorylation controls remodeling of the actin cytoskeleton.

Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with ;locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.

SMILE, Topography-Guided LASIK, and Wavefront-Guided LASIK: Review of Clinical Outcomes in Premarket Approval FDA Studies.

PURPOSE: To review clinical outcomes of the U.S. Food and Drug Administration premarket approval (PMA) studies of the three recently approved refractive surgery platforms for the correction of spherocylindrical myopia. METHODS: Clinical outcomes and vector analysis of astigmatism were reviewed and compared between wavefront-guided laser in situ keratomileusis (LASIK) (WFG group) (STAR S4 IR with iDesign aberrometer; Johnson & Johnson Vision Care, Inc., Santa Ana, CA), topography-guided LASIK (TOPO group) (Allegretto Wave Eye-Q laser; Alcon Laboratories, Inc., Fort Worth, TX), and small incision lenticule extraction (SMILE group) (VisuMax laser; Carl Zeiss Meditec AG, Jena, Germany). RESULTS: The number of eyes included was 304, 249, and 334 for the SMILE, TOPO, and WFG groups, respectively. The WFG group had the highest preoperative spherical equivalent refraction (-6.21 ± 2.78 diopters [D]), followed by the SMILE (-5.39 ± 2.30 D) and TOPO (-4.61 ± 2.43 D) groups. At 6 months postoperatively, 83.7%, 88.9%, and 82.6% of eyes in the SMILE, TOPO, and WFG groups, respectively, had uncorrected distance visual acuity (UDVA) of 20/20 or better. SMILE showed slower visual recovery at early postoperative examinations, whereas the WFG group had a decrease in UDVA over time due to refractive regression. The mean refractive cylinder changed from -1.53 ± 0.70 D preoperatively to -0.22 ± 0.33 D at 6 months postoperatively in the SMILE group, -1.19 ± 1.23 to -0.19 ± 0.30 D in the TOPO group, and -1.77 ± 1.65 to -0.33 ± 0.36 D in the WFG group. With all three procedures, undercorrection of refractive cylinder was seen with increasing attempted correction. CONCLUSIONS: All three procedures demonstrated excellent outcomes, considering differences in attempted correction between studies. No obvious superiority of one technique over the others was found in astigmatic correction. [J Refract Surg. 2019;35(11):690-698.].

Analyzing Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Aging and Vascular Amyloid Pathology.

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive [Formula: see text] which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age-dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein.

Impaired melanoma growth in VASP deficient mice.

Progression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role of VASP for melanoma growth in murine allograft models. Growth of VASP expressing melanomas was retarded in VASP(-/-) versus wild-type animals. Over time tumor size was <50% in VASP(-/-) versus wild-type animals and independent of expression levels of Ena/VASP protein family members. Histological analyses showed smaller cells with impaired nutrition status and less vascularization in melanomas derived from VASP(-/-) versus counterparts from wild-type mice. Cumulatively, the data reveal a critical role of VASP in non-tumor cells in the tumor environment for melanoma growth in vivo.

Transmitter- and hormone-activated Ca(2+) responses in adult microglia/brain macrophages in situ recorded after viral transduction of a recombinant Ca(2+) sensor.

In vitro studies show that microglia, the resident immune cells of the brain, express neurotransmitter and neuropeptide receptors which are linked to Ca(2+) signaling. Here we describe an approach to obtain Ca(2+) recordings from microglia in situ. We injected a retrovirus encoding a calcium sensor into the cortex of mice 2 days after stimulation of microglial proliferation by a stab wound injury. Microglial cells were identified with tomato lectin in acute slices prepared 3, 6, 21 and 42 days after the injury. The membrane current profile and the ameboid morphology indicated that microglial cells were activated at day 6 while at day 42 they resembled resting microglia. We recorded transient Ca(2+) responses to application of ATP, endothelin-1, substance P, histamine and serotonin. The fluorescence amplitude of ATP was increased only at day 6 compared to other time points, while responses to all other ligands did not vary. Only half of the microglial cells that responded to ATP also responded to endothelin-1, serotonin and histamine. Substance P, in contrast, showed a complete overlap with the ATP responding microglial population at day 6, at day 42 this population was reduced to 55%. Cultured cells were less responsive to these ligands. This study shows that in situ microglia consist of heterogeneous populations with respect to their sensitivity to neuropeptides and -transmitters.

Trypsin induces epidermal proliferation and inflammation in murine skin.

Human keratinocytes are known to express the protease-activated receptors, PAR-1 and PAR-2. Activation of PAR-1 results in increased proliferation, whereas PAR-2 activation results in decreased keratinocyte proliferation. Trypsin activates PAR-1 and in higher concentrations, PAR-2. The aim of this study was to evaluate the overall effect of trypsin on keratinocyte proliferation in a mouse in vivo and in vitro model. Daily topical application of 0.3-300 pmol trypsin/cm2 on hairless mouse skin induced dose-dependent epidermal hyperproliferation as determined by an increase in 5-bromo-2'-deoxyuridine incorporation of up to eight-fold in basal keratinocytes and an up to three-fold increase in keratinocyte layers. This was accompanied by an increased transepidermal water loss. These effects of trypsin were abolished by the addition of the trypsin inhibitor n-p-tosyl-l-lysine-chloromethyl ketone. Histological analysis revealed acanthosis, hypergranulosis, and spongiosis in the epidermis as well as vasodilatation and an inflammatory infiltrate in the upper dermis. In the murine keratinocyte cell line PAM-212 activation of PAR-1 with specific activating peptides resulted in a calcium influx and an increase of proliferation, whereas activation of PAR-2 caused a diminished proliferation. Incubation with trypsin, PAR-1-, and PAR-2-activating peptides induced cytokine-induced neutrophil chemoattractant (KC) mRNA expression as a marker for inflammation in PAM-212 in a dose-dependent manner. In conclusion, our results suggest that trypsin induces in vivo epidermal proliferation and inflammation. Proliferation seems not to be signaled by PAR activation, but PAR-2-induced KC chemokine expression may contribute in part to trypsin-induced inflammation.