Ketamine modulates disrupted in schizophrenia-1/glycogen synthase kinase-3ß interaction.

Introduction: Disrupted in schizophrenia-1 (DISC1) is a scaffolding protein whose mutated form has been linked to schizophrenia, bipolar affective disorders, and recurrent major depression. DISC1 regulates multiple signaling pathways involved in neurite outgrowth and cortical development and binds directly to glycogen synthase kinase-3ß (GSK-3ß). Since ketamine activates GSK-3ß, we examined the impact of ketamine on DISC1 and GSK-3ß expression. Methods: Postnatal day 7 rat pups were treated with ketamine with and without the non-specific GSK-3ß antagonist, lithium. Cleaved-caspase-3, GSK-3ß and DISC1 levels were measured by immunoblots and DISC1 co-localization in neurons by immunofluorescence. Binding of DISC1 to GSK-3ß was determined by co-immunoprecipitation. Neurite outgrowth was determined by measuring dendrite and axon length in primary neuronal cell cultures treated with ketamine and lithium. Results: Ketamine decreased DISC1 in a dose and time-dependent manner. This corresponded to decreases in phosphorylated GSK-3ß, which implicates increased GSK-3ß activity. Lithium significantly attenuated ketamine-induced decrease in DISC1 levels. Ketamine decreased co-immunoprecipitation of DISC1 with GSK-3ß and axonal length. Conclusion: These findings confirmed that acute administration of ketamine decreases in DISC1 levels and axonal growth. Lithium reversed this effect. This interaction provides a link between DISC1 and ketamine-induced neurodegeneration.

Mechanism of isoflurane­mediated breast cancer growth in vivo.

Use of volatile anesthetics is associated with worse outcome following tumor resection surgery compared with the use of intravenous anesthetics. However, the underlying mechanism has not been clearly delineated yet in vivo. The EO771 cell-based congenic breast cancer model was used in the present study. Isoflurane directly binds to and inhibits two adhesion molecules, leukocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1). Similarly, exposure to sevoflurane, another volatile anesthetic and LFA-1 inhibitor, is associated with an increase in breast cancer size compared with non-exposure. Thus, the present study first examined the role of LFA-1 and Mac-1 in the EO771 breast cancer model. Both LFA-1 deficiency and inhibition enhanced tumor growth, which was supported by cytokine and eicosanoid data profiles. By contrast, Mac-1 deficiency did not affect tumor growth. The exposure to isoflurane and sevoflurane was associated with an increase in breast cancer size compared with non-exposure. These data suggested that isoflurane enhanced tumor growth by interacting with LFA-1. Isoflurane exposure did not affect tumor growth in LFA-1-deficient mice. In summary, the present data showed that LFA-1 deficiency facilitated breast cancer growth, and isoflurane, an LFA-1 inhibitor, also increased breast cancer growth.

The landscape of immune dysregulation in pediatric sepsis at a single-cell resolution.

Recognizing immune dysregulation as a hallmark of sepsis pathophysiology, leukocytes have attracted major attention of investigation. While adult and pediatric sepsis are clinically distinct, their immunological delineation remains limited. Single cell technologies facilitated the characterization of immune signatures. We tackled to delineate immunological profiles of pediatric sepsis at a single-cell level by analyzing blood samples from six septic children, at both acute and recovery phases, and four healthy children. 16 single-cell transcriptomic datasets were analyzed and compared to adult sepsis dataset. We showed a unique shift in neutrophil subpopulations and functions between acute and recovery phases, along with the regulatory role of resistin. Neutrophil signatures were comparable between adult and pediatric sepsis. Innate-like CD4 T cells were predominantly and uniquely observed in acute phase of pediatric sepsis. Our study serves as a rich source of information about the phenotypic diversity and trajectory of circulating immune cells during pediatric sepsis.

Integrin CD11c regulates B cell homeostasis.

CD11c is widely known as a cell surface marker for dendritic cells, but we recently showed that it regulates neutrophil and T cell functions. Because we found that CD11c knockout (KO) mice had lower blood B cell counts, we characterized B cell profile in developmental stages. We found that CD11c KO recirculating and mature B cells was significantly fewer compared with wild type, associated with exaggerated proliferation and apoptosis. Because they did not express CD11c, we sought for the possibility of CD11c-mediated non-intrinsic regulation of B cell proliferation and apoptosis. Here we hypothesized that dendritic cells, major cells expressing CD11c would regulate B cells indirectly. The proteomics of dendritic cells cultured in vitro indicated the downregulation of macrophage migration inhibitory factor (MIF). Less MIF was also confirmed by ELISA. Furthermore, plasma MIF level was significantly lower in naïve CD11c KO mice. Because MIF regulates B cell survival, we demonstrated a novel regulatory mechanism of naïve B cells via CD11c.

Cystic Fibrosis Mice Are Highly Susceptible to Repeated Acute Pseudomonas aeruginosa Pneumonia after Intranasal Inoculation.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that controls chloride current. A number of different CFTR transgenic mouse lines have been developed and subjected to both acute and chronic infection models. However, prior studies showed no substantial differences in bacterial clearance between CF and non-CF mice after single inoculations. Here, using F508del transgenic CF mice, we examined the role of repeated acute Pseudomonas aeruginosa (PA) infection, with the second inoculation 7 days after the first. We found that CF mice were more susceptible to PA infection than non-CF mice following the second inoculation, with non-CF mice showing better neutrophil recruitment and effector functions. We further investigated the characteristics of lung immune cells using single-cell RNA sequencing, finding that non-CF lung neutrophils had more prominent upregulation of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) compared to CF lung neutrophils. Although people with CF are often colonized with bacteria and have high numbers of neutrophils in the airways during chronic infection, these data suggest that CF neutrophils have deficient effector functions in the setting of repeated acute infection.

Perioperative Factors and Radiographic Severity Scores for Predicting the Duration of Mechanical Ventilation After Arterial Switch Surgery.

OBJECTIVES: Cardiac surgery on cardiopulmonary bypass (CPB) during the neonatal period can cause perioperative organ injuries. The primary aim of this study was to determine the incidence and risk factors associated with postoperative mechanical ventilation duration and acute lung injury after the arterial switch operation (ASO). The secondary aim was to examine the utility of the Brixia score for characterizing postoperative acute lung injury (ALI). DESIGN: A retrospective study. SETTING: A single-center university hospital. PARTICIPANTS: A total of 93 neonates with transposition of great arteries with intact ventricular septum (dTGA IVS) underwent ASO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: From January 2015 to December 2022, 93 neonates with dTGA IVS were included in the study. The cohort had a median age of 4.0 (3.0-5.0) days and a mean weight of 3.3 ± 0.5 kg. About 63% of patients had ≥48 hours of postoperative mechanical ventilation after ASO. Risk factors included prematurity, post-CPB transfusion of salvaged red cells, platelets and cryoprecipitate, and postoperative fluid balance by univariate analysis. The larger transfused platelet volume was associated with the risk of ALI by multivariate analysis. The median baseline Brixia scores were 11.0 (9.0-12.0) and increased significantly in the postoperative day 1 in patients who developed moderate ALI 24 hours after admission to the intensive care unit (15.0 [13.0-16.0] v 12.0 [10.0-14.0], p = 0.046). CONCLUSIONS: Arterial switch operation results in a high incidence of ≥48-hour postoperative mechanical ventilation. Blood component transfusion is a potentially modifiable risk factor. The Brixia scores also may be used to characterize postoperative acute lung injury.

The landscape of immune dysregulation in pediatric sepsis at a single-cell resolution.

Recognizing immune dysregulation as a hallmark of sepsis pathophysiology, leukocytes have attracted major attention of investigation. While adult and pediatric sepsis are clinically distinct, their immunological delineation remains limited. Breakthrough of single cell technologies facilitated the characterization of immune signatures. We tackled to delineate immunological profiles of pediatric sepsis at a single-cell level by analyzing blood samples from six septic children, at both acute and recovery phases, and four healthy children. 16 single-cell transcriptomic datasets (96,156 cells) were analyzed and compared to adult sepsis dataset. We showed a unique shift in neutrophil subpopulations and functions between acute and recovery phases, along with examining the regulatory role of resistin. Neutrophil signatures were comparable between adult and pediatric sepsis. Innate-like CD4 T cells were predominantly and uniquely observed in acute phase of pediatric sepsis. Our study provides a thorough and comprehensive understanding of immune dysregulation in pediatric sepsis.

CD11a regulates hematopoietic stem and progenitor cells.

Integrin αLß2 (CD11a/CD18, CD11a) is a critical leukocyte adhesion molecule in leukocyte arrest and immunological synapse formation. However, its role in the bone marrow has not been investigated in depth. Here we showed that CD11a was expressed on all subsets of hematopoietic stem and progenitor cells (HPSCs). CD11a deficiency enhanced HSPCs activity under lipopolysaccharide (LPS) stimulation as demonstrated by a higher HSPC cell count along with an increase in cell proliferation. However, our mixed chimera experiment did not support that this phenotype was driven in a cell-intrinsic manner. Rather we found that the production of IL-27, a major cytokine that drives HSPC proliferation, was significantly upregulated both in vivo and in vitro. This adds a novel role of CD11a biology.

Narrative review of systemic inflammatory response mechanisms in cardiac surgery and immunomodulatory role of anesthetic agents.

Although surgical techniques and perioperative care have made significant advances, perioperative mortality in cardiac surgery remains relatively high. Single- or multiple-organ failure remains the leading cause of postoperative mortality. Systemic inflammatory response syndrome (SIRS) is a common trigger for organ injury or dysfunction in surgical patients. Cardiac surgery involves major surgical dissection, the use of cardiopulmonary bypass (CPB), and frequent blood transfusions. Ischemia-reperfusion injury and contact activation from CPB are among the major triggers for SIRS. Blood transfusion can also induce proinflammatory responses. Here, we review the immunological mechanisms of organ injury and the role of anesthetic regimens in cardiac surgery.

Perioperative prophylactic corticosteroids for cardiac surgery in children: A systematic review and meta-analysis.

OBJECTIVE: Perioperative corticosteroids have been used for pediatric cardiac surgery for decades, but the underlying evidence is conflicting. We aimed to investigate the efficacy and safety of perioperative prophylactic corticosteroids in pediatric heart surgeries. METHODS: We searched electronic databases until March 2023 to retrieve all randomized controlled trials (RCTs) that administered perioperative prophylactic corticosteroids to children undergoing heart surgery. We used RevMan 5.4 to pool risk ratios (RRs) and mean differences (MDs). RESULTS: A total of 12 RCTs (2,209 patients) were included in our review. Corticosteroids administration was associated with a nonsignificant reduction in all-cause mortality (RR 0.62; 95% CI: 0.37-1.02, I2 = 0%; moderate certainty); however, it was associated with a lower duration of mechanical ventilation (MV) (MD -0.63 days; 95% CI: -1.16 to -0.09 days, I2 = 41%; high certainty). Corticosteroids did not affect the length of ICU and hospital stay but significantly reduced the incidence of postoperative low cardiac output syndrome (LCOS) (RR 0.76; 95% CI: 0.60-0.96, I2 = 0%; moderate certainty) and reoperation (RR 0.37; 95% CI: 0.19-0.74, I2 = 0%; moderate certainty). There was no increase in adverse events except a higher risk of hyperglycemia and postoperative insulin use. CONCLUSIONS: The use of perioperative corticosteroids in pediatric heart surgeries is associated with a trend toward reduced all-cause mortality without attaining statistical significance. Corticosteroids reduced MV duration, and probably decrease the incidence of LCOS, and reoperations. The choice of corticosteroid agent and dose is highly variable and further larger studies may help determine the ideal agent, dose, and patient population for this prophylactic therapy.

Volatile anesthetic isoflurane exposure facilitates Enterococcus biofilm infection.

Enterococcus faecalis (E. faecalis) is one of the major pathogenic bacteria responsible for surgical site infections. Biofilm infections are major hospital-acquired infections. Previous studies suggested that ions could regulate biofilm formation in microbes. Volatile anesthetics, frequently administered in surgical setting, target ion channels. Here, we investigated the role of ion channels/transporters and volatile anesthetics in the biofilm formation by E. faecalis MMH594 strain and its ion transporter mutants. We found that a chloride transporter mutant significantly reduced biofilm formation compared to the parental strain. Downregulation of teichoic acid biosynthesis in the chloride transporter mutant impaired biofilm matrix formation and cellular adhesion, leading to mitigated biofilm formation. Among anesthetics, isoflurane exposure enhanced biofilm formation in vitro and in vivo. The upregulation of de novo purine biosynthesis pathway by isoflurane exposure potentially enhanced biofilm formation, an essential process for DNA, RNA, and ATP synthesis. We also demonstrated that isoflurane exposure to E. faecalis increased cyclic-di-AMP and extracellular DNA production, consistent with the increased purine biosynthesis. We further showed that isoflurane enhanced the enzymatic activity of phosphoribosyl pyrophosphate synthetase (PRPP-S). With the hypothesis that isoflurane directly bound to PRPP-S, we predicted isoflurane binding site on it using rigid docking. Our study provides a better understanding of the underlying mechanisms of E. faecalis biofilm formation and highlights the potential impact of an ion transporter and volatile anesthetic on this process. These findings may lead to the development of novel strategies for preventing E. faecalis biofilm formation and improving patient outcomes in clinical settings.

Predictability of Pediatric Sepsis Outcome Using SEPSIS-3 Definition in a Single Tertiary Pediatric Institution.

Sepsis is a syndrome of dysregulated response to infection and is associated with high morbidity and mortality. Sepsis was initially defined as a host's systemic inflammatory response syndrome (SIRS) to infection. In 2016, the importance of dysregulated response was incorporated into the definition of sepsis; adult sepsis was redefined as a life-threatening organ dysfunction caused by a dysregulated host response to infection, with organ function being evaluated by the Sequential Organ Failure Assessment (SOFA) score (Sepsis-3 definition). However, the definition of pediatric sepsis remains the same, based on the original, SIRS-based criteria. In this study, we examined the relationship between mortality and sepsis in pediatric patients in our institution using the Sepsis-3 definition by incorporating the pediatric SOFA (pSOFA) score system, which was reported in 2017. We found that sepsis mortality was better correlated with the pSOFA score in our pediatric cohort. We also found that patients who did not have identified microbes were associated with better survival. In the future, we need to determine the relationship between mortality and Sepsis-3 definition-based pediatric sepsis worldwide to further define the utility of this new definition.

Age-dependent transcriptomic profiles of leukocytes in pediatric population.

Immunity at birth is considered immature. Following birth, our immune function is considered to grow and reach maturation over time. To obtain granular information of leukocyte functions and transcriptomic profiles in pediatric cohort, we examined leukocyte profiles in infants, preschool and school children using single cell RNA sequencing of their peripheral blood mononuclear cells (PBMCs). Monocytes and natural killer (NK) cells showed immaturity in infants. Their innate and adaptive immunity was developed by preschool age. Adaptive immune cells showed different maturation patterns. CD4, CD8 naïve T cells and plasma cells continued to mature untill school age. In CD8 naïve T cells, innate immunity was upregulated in infants, in support of our knowledge that they manifests more innate cell-like phenotype soon after birth. Many signaling pathways have been differentially up- and/or down-regulated in infants, preschool and school children. Their contribution to the development of the immune system needs to be delineated.

Differential effects of dexmedetomidine on Gram-positive and Gram-negative bacterial killing and phagocytosis.

Dexmedetomidine is a commonly used sedative in perioperative and intensive care settings with purported immunomodulatory properties. Since its effects on immune functions against infections have not been extensively studied, we tested the effects of dexmedetomidine on Gram-positive [Staphylococcus aureus and Enterococcus faecalis] and Gram-negative bacteria [Escherichia coli], and on effector functions of human monocytes THP-1 cells against them. We evaluated phagocytosis, reactive oxygen species (ROS) formation, and CD11b activation, and performed RNA sequencing analyses. Our study revealed that dexmedetomidine improved Gram-positive but mitigated Gram-negative bacterial phagocytosis and killing in THP-1 cells. The attenuation of Toll-like receptor 4 (TLR4) signaling by dexmedetomidine was previously reported. Thus, we tested TLR4 inhibitor TAK242. Similar to dexmedetomidine, TAK242 reduced E. coli phagocytosis but enhanced CD11b activation. The reduced TLR4 response potentially increases CD11b activation and ROS generation and subsequently enhances Gram-positive bacterial killing. Conversely, dexmedetomidine may inhibit the TLR4-signaling pathway and mitigate the alternative phagocytosis pathway induced by TLR4 activation through LPS-mediated Gram-negative bacteria, resulting in worsened bacterial loads. We also examined another α2 adrenergic agonist, xylazine. Because xylazine did not affect bacterial clearance, we proposed that dexmedetomidine may have an off-target effect on bacterial killing process, potentially involving crosstalk between CD11b and TLR4. Despite its potential to attenuate inflammation, we provide a novel insight into potential risks of dexmedetomidine use during Gram-negative infections, highlighting the differential effect of dexmedetomidine on Gram-positive and Gram-negative bacteria.

Surgical site infection in pediatric spinal fusion surgery revisited: outcome and risk factors after preventive bundle implementation.

Background: Surgical site infections (SSI) contribute to significant morbidity, mortality, length of stay, and financial burden. We sought to evaluate the incidence and risk factors of surgical site infection following pediatric spinal fusion surgery in patients for whom standard perioperative antibiotic prophylaxis and preventive strategies have been implemented. Methods: We conducted a retrospective study of children aged <18 years who underwent spinal fusion surgery from January 2017 to November 2021 at a quaternary academic pediatric medical center. Univariable analysis was used to evaluate associations between potential risk factors and SSI. Results: Of 1111 patients, 752 (67.6%) were female; median age was 14.2 years. SSI occurred in 14 patients (1.3%). Infections were superficial incisional (n=2; 14.3%), deep incisional (n=9; 64.3%), and organ/space (n=3; 21.4%). Median time to SSI was 14 days (range, 8 to 45 days). Staphylococcus aureus and Escherichia coli were the most frequently-isolated bacteria. Potential risk factors for SSIs included low body weight (Odds ratio (OR) 0.96, 95% confidence interval (CI) 0.93-0.99, p=0.026), ASA classification of ≥3 (OR 24.53, 95%CI 3.20-188.22, p=0.002), neuromuscular scoliosis (OR 3.83, 95%CI 3.82-78.32, p<0.001), prolonged operative time (OR 1.56, 95%CI 1.28-1.92, p<0.001), prolonged anesthetic time (OR 1.65, 95%CI 1.35-2.00, p<0.001), administration of prophylactic antibiotic ≥60 minutes before skin incision (OR 11.52, 95%CI 2.34-56.60, p=0.003), and use of povidone-iodine alone for skin preparation (OR 5.97, 95%CI 1.27-28.06, p=0.024). Conclusion: In the context of a robust bundle for SSI prevention; low body weight, ASA classification of ≥3, neuromuscular scoliosis, prolonged operative and anesthetic times, administration of prophylactic antibiotic ≥60 minutes before skin incision, and use of povidone-iodine alone for skin preparation increased the risk of SSI. Administration of prophylactic antibiotic within 60 minutes of skin incision, strict adherence to high-risk preventive protocol, and use of CHG-alcohol could potentially reduce the rate of SSI.

CD11c regulates neutrophil maturation.

Sepsis continues to be associated with high morbidity and mortality. Currently, sepsis is managed only conservatively. In sepsis, a substantial number of neutrophils is required, leading to accelerated neutrophil production. Immature neutrophils are released into the circulation to meet a demand, despite their less effective functioning in microbial eradication. Although an intervention to provide more mature neutrophils may serve as a potential sepsis treatment, the mechanism of neutrophil differentiation and maturation remains poorly understood. We discovered that CD11c, traditionally known as a dendritic cell marker, was expressed in neutrophils and regulated neutrophil maturation and effector functions. In the absence of CD11c, neutrophil maturation was impaired in the bone marrow, concomitant with a significant increase in the proliferation and apoptosis of preneutrophils, associated with less effector functions. Under lipopolysaccharide challenge, inducing an emergent neutrophil production in the bone marrow, CD11c deficiency exaggerated the release of immature neutrophils into the circulation, associated with a significant proliferation and apoptosis of preneutrophils. In contrast, constitutively active CD11c knock-in mice showed accelerated neutrophil maturation associated with enhanced effector functions, which further supports the notion that CD11c regulates neutrophil maturation. Furthermore, the constitutively active CD11c knock-in mice offered enhanced bacterial eradication. Taken together, we discovered that CD11c was critical for the regulation of neutrophil maturation, and CD11c activation could serve as a potential target for sepsis treatment.

Neutrophil extracellular traps formation is associated with postoperative complications in neonates and infants undergoing congenital cardiac surgery.

Pediatric patients with congenital heart diseases (CHD) often undergo surgical repair on cardiopulmonary bypass (CPB). Despite a significant medical and surgical improvement, the mortality of neonates and infants remains high. Damage-associated molecular patterns (DAMPs) are endogenous molecules released from injured/damaged tissues as danger signals. We examined 101 pediatric patients who underwent congenital cardiac surgery on CPB. The mortality rate was 4.0%, and the complication rate was 31.6%. We found that neonates/infants experienced multiple complications most, consistent with the previous knowledge. Neonates and infants in the complication group had received more transfusion intraoperatively than the non-complication arm with lower maximum amplitude (MA) on rewarming CPB thromboelastography (TEG). Despite TEG profiles were comparable at ICU admission between the two groups, the complication arm had higher postoperative chest tube output, requiring more blood transfusion. The complication group showed greater neutrophil extracellular traps (NETs) formation at the end of CPB and postoperatively. Plasma histones and high mobility group box 1 (HMGB1) levels were significantly higher in the complication arm. Both induced NETs in vitro and in vivo . As histones and HMGB1 target Toll-like receptor (TLR)2 and TLR4, their mRNA expression in neutrophils was upregulated in the complication arm. Taken together, NETs play a major role in postoperative complication in pediatric cardiac surgery and would be considered a target for intervention. Key points: Neonates and infants showed highest postoperative complications with more upregulation of inflammatory transcriptomes of neutrophils.Neonates and infants with organ dysfunction had more NETs formation with higher plasma histones and HMGB1 levels.

αDß2 as a novel target of experimental polymicrobial sepsis.

Since sepsis was defined three decades ago, it has been a target of intensive study. However, there is no specific sepsis treatment available, with its high mortality and morbidity. αDß2 (CD11d/CD18) is one of the four ß2 integrin members. Its role in sepsis has been limitedly studied. Using an experimental polymicrobial sepsis model, we found that the deficiency of αDß2 was associated with less lung injury and better outcome, which was in sharp contrast to other ß2 integrin member αLß2 (CD11a/CD18), and αMß2 (CD11b/CD18). This phenotype was supported by a reduction of bacterial loads in αDß2 knockout mice. Further analysis showed that the deficiency of αDß2 led to a reduction of neutrophil cell death as well as an increase in neutrophil phagocytosis in both murine and human systems. Our data showed a unique role of αDß2 among the ß2 integrin members, which would serve as a potential target to improve the outcome of sepsis.

GltS regulates biofilm formation in methicillin-resistant Staphylococcus aureus.

Biofilm-based infection is a major healthcare burden. Methicillin-resistant Staphylococcus aureus (MRSA) is one of major organisms responsible for biofilm infection. Although biofilm is induced by a number of environmental signals, the molecule responsible for environmental sensing is not well delineated. Here we examined the role of ion transporters in biofilm formation and found that the sodium-glutamate transporter gltS played an important role in biofilm formation in MRSA. This was shown by gltS transposon mutant as well as its complementation. The lack of exogenous glutamate also enhanced biofilm formation in JE2 strain. The deficiency of exogenous glutamate intake accelerated endogenous glutamate/glutamine production, which led to the activation of the urea cycle. We also showed that urea cycle activation was critical for biofilm formation. In conclusion, we showed that gltS was a critical regulator of biofilm formation by controlling the intake of exogenous glutamate. An intervention to target glutamate intake may be a potential useful approach against biofilm.