Therapeutic Effects of Hyaluronic Acid Against Cytotoxic Extracellular Vesicles Released During Pseudomonas Aeruginosa Pneumonia.

ABSTRACT: Extracellular vesicles (EVs) have now been recognized as important mediators of cellular communication during injury and repair. We previously found that plasma EVs isolated from ex vivo perfused human lungs injured with Escherichia coli bacterial pneumonia were inflammatory, and exogenous administration of high molecular weight (HMW) hyaluronic acid (HA) as therapy bound to these EVs, decreasing inflammation and injury. In the current study, we studied the role of EVs released during severe Pseudomonas aeruginosa (PA) pneumonia in mice and determined whether intravenous administration of exogenous HMW HA would have therapeutic effects against the bacterial pneumonia. EVs were collected from the bronchoalveolar lavage fluid (BALF) of mice infected with PA103 by ultracentrifugation and analyzed by NanoSight and flow cytometry. In a cytotoxicity assay, administration of EVs released from infected mice (I-EVs) decreased the viability of A549 cells compared to EV isolated from sham control mice (C-EVs). Either exogenous HMW HA or an anti-CD44 antibody, when co-incubated with I-EVs, significantly improved the viability of the A549 cells. In mice with PA103 pneumonia, administration of HMW HA improved pulmonary edema and bacterial count in the lungs and decreased TNF-α and caspase-3 levels in the supernatant of lung homogenates. In conclusion, EVs isolated from BALF of mice with P. aeruginosa pneumonia were cytotoxic and inflammatory, and intravenous HMW HA administration was protective against P. aeruginosa pneumonia.

Epidemiological survey of serum titers from adults against various Gram-negative bacterial V-antigens.

The V-antigen, a virulence-associated protein, was first identified in Yersinia pestis more than half a century ago. Since then, other V-antigen homologs and orthologs have been discovered and are now considered as critical molecules for the toxic effects mediated by the type III secretion system during infections caused by various pathogenic Gram-negative bacteria. After purifying recombinant V-antigen proteins, including PcrV from Pseudomonas aeruginosa, LcrV from Yersinia, LssV from Photorhabdus luminescens, AcrV from Aeromonas salmonicida, and VcrV from Vibrio parahaemolyticus, we developed an enzyme-linked immunoabsorbent assay to measure titers against each V-antigen in sera collected from 186 adult volunteers. Different titer-specific correlation levels were determined for the five V-antigens. The anti-LcrV and anti-AcrV titers shared the highest correlation with each other with a correlation coefficient of 0.84. The next highest correlation coefficient was between anti-AcrV and anti-VcrV titers at 0.79, while the lowest correlation was found between anti-LcrV and anti-VcrV titers, which were still higher than 0.7. Sera from mice immunized with one of the five recombinant V-antigens displayed cross-antigenicity with some of the other four V-antigens, supporting the results from the human sera. Thus, the serum anti-V-antigen titer measurement system may be used for epidemiological investigations of various pathogenic Gram-negative bacteria.

Therapeutic Effects of Hyaluronic Acid in Peritonitis-Induced Sepsis in Mice.

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 µg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Therapeutic Effects of Hyaluronic Acid in Bacterial Pneumonia in Ex Vivo Perfused Human Lungs.

Rationale: Recent studies have demonstrated that extracellular vesicles (EVs) released during acute lung injury (ALI) were inflammatory.Objectives: The current study was undertaken to test the role of EVs induced and released from severe Escherichia coli pneumonia (E. coli EVs) in the pathogenesis of ALI and to determine whether high-molecular-weight (HMW) hyaluronic acid (HA) administration would suppress lung injury from E. coli EVs or bacterial pneumonia.Methods:E. coli EVs were collected from the perfusate of an ex vivo perfused human lung injured with intrabronchial E. coli bacteria for 6 hours by ultracentrifugation and then given intrabronchially or intravenously to naive human lungs. One hour later, HMW HA was instilled into the perfusate (n = 5-6). In separate experiments, HMW HA was given after E. coli bacterial pneumonia (n = 6-10). In vitro experiments were conducted to evaluate binding of EVs to HMW HA and uptake of EVs by human monocytes.Measurements and Main Results: Administration of HMW HA ameliorated the impairment of alveolar fluid clearance, protein permeability, and acute inflammation from E. coli EVs or pneumonia and reduced total bacteria counts after E. coli pneumonia. HMW HA bound to E. coli EVs, inhibiting the uptake of EVs by human monocytes, an effect associated with reduced TNFα (tumor necrosis factor α) secretion. Surprisingly, HMW HA increased E. coli bacteria phagocytosis by monocytes.Conclusions: EVs induced and released during severe bacterial pneumonia were inflammatory and induced ALI, and HMW HA administration was effective in inhibiting the uptake of EVs by target cells and decreasing lung injury from E. coli EVs or bacterial pneumonia.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Decrease Lung Injury in Mice.

Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B4 in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB4 BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB4 and LTC4 from LTA4 are competitive, and MRP1 is the efflux pump for LTC4 Inhibition of MRP1 will increase LTB4 production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC4 and subsequently increased LTB4 levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB4 production and antimicrobial activity through LTB4/BLT1 signaling.

Anesthetic management of a pediatric patient with arginase-1 deficiency undergoing strabismus operation: a case report.

BACKGROUND: Urea cycle disorders are rare; arginase-1 deficiency is one of those extremely rare autosomal recessive metabolic disorders. Arginase-1 is one among the enzymes involved in the production of urea from ammonia in the liver, and its deficiency produces the characteristic feature, hyperargininemia. CASE PRESENTATION: We report a case of a girl, aged 5 years and 10 months presenting with arginase-1 deficiency. The patient was scheduled to undergo strabismus surgery for intermittent exotropia under general anesthesia. Preoperative blood tests showed high serum arginine levels, but ammonia levels were within the normal range. Anesthesia was induced with sevoflurane and nitrous oxide via face mask and maintained with sevoflurane, fentanyl, and rocuronium. Vital signs were stable throughout the surgery. There was an intraoperative decrease in blood glucose levels (from 82 mg/dL to 42 mg/dL) that was treated with intravenous glucose. Arginine levels remained high after surgery; however, hyperammonemia did not develop. There were no complications and the patient was discharged on the following day. CONCLUSIONS: We successfully performed general anesthesia in a child with hyperargininemia. Only a few cases of arginase-1 deficiency had been reported and much remains unknown about its pathology. Therefore, information sharing among medical professionals is essential to customize the plan for the management of this disorder in patients.

An outbreak of fluoroquinolone-resistant Pseudomonas aeruginosa ST357 harboring the exoU gene.

Antimicrobial-resistant isolates of Pseudomonas aeruginosa collected from 2005 to 2014 in a university hospital in Kyoto, Japan, were retrospectively analyzed by multilocus sequence typing (MLST), exoenzyme genotype determination, integron characterization, and clinical associations. During the study, 1573 P. aeruginosa isolates were detected, and 41 of these were resistant to more than two classes of antimicrobial agents. Twenty-five (61.0%) isolates were collected from urine. All isolates were resistant to ciprofloxacin, 8 (19.5%) isolates showed resistance to imipenem/cilastatin, and 8 (19.5%) isolates showed resistance to meropenem. None of the isolates fulfilled the clinical criteria for multidrug-resistant P. aeruginosa. All isolates were negative in the metallo-ß lactamase test. Thirty-six (87.8%) isolates were of the exoS-exoU+ genotype and 5 (12.2%) isolates were of the exoS+exoU- genotype. Among 36 exoS-exoU+ isolates, 33 (80.5%) were ST357, and 3 (7.3%) were ST235. Five isolates of exoS+exoU- were ST186, ST244, ST314, ST508, and ST512. Thirty-three isolates were positive for class 1 integrons and four different class 1 integrons were detected: aminoglycoside (2') adenyltransferase and chloramphenicol transporter (AadB+CmlA6), OXA-4 ß-lactamase and aminoglycoside 3'-adenyltransferase (OXA4+AadA2), AadB alone, and aminoglycoside acetyltransferase alone (AacA31). Among the 41 patients from which the isolates originated, the most common underlying disease was cancer in 16 patients (39%), and 9 patients (22.0%) died during the hospitalization period. There was no statistical correlation between MLST, exoenzyme genotype, and patient mortality. The results indicated outbreaks of fluoroquinolone-resistant P. aeruginosa in immunocompromised patients mainly due to the propagation of potentially virulent ST357 isolates possessing the exoU+ genotype.

Flow-dynamics assessment of mitral-valve surgery by intraoperative vector flow mapping.

OBJECTIVES: We assessed vortex patterns and energy loss in left ventricular flow in patients who underwent mitral valve repair or replacement with bioprosthetic valves. METHODS: Vector flow mapping was performed before and after the procedure in 15 and 17 patients who underwent repair and replacement, respectively. The preprocedure mitral-septal angle was measured in all patients. Relationships between vortex patterns or energy loss change (ELC) and annuloplasty ring or bioprosthetic valve sizes or the effect of mitral leaflet resection in the repair group were statistically analysed. RESULTS: Normal vortex patterns were observed in 13 and 1 patients who underwent repair and replacement, respectively. Abnormal vortex patterns were observed in 2 and 16 patients who underwent repair and replacement, respectively. ELC was significantly higher in the replacement group (196.6 ± 180.8) than in the repair group (71.9 ± 43.9). In the repair group, preoperative mitral-septal angles in patients with normal vortex patterns (79.2° ± 3.4°) were significantly larger than those in patients with abnormal vortex patterns (67.5° ± 3.5°). No significant differences were observed in the effects of annuloplasty ring and bioprosthetic valve sizes on vortex patterns and ELC, and in the effect of mitral valve resection (80.4 ± 56.3) and respect (without leaflet resection) (53.8 ± 28.4) on ELC in the repair group. CONCLUSIONS: Mitral valve replacement alters the intraventricular vortex pattern and increases flow energy loss. A small mitral-septal angle is a risk factor for abnormal vortex patterns after mitral valve repair surgery.

Vector flow mapping analysis of left ventricular energetic performance in healthy adult volunteers.

BACKGROUND: Vector flow mapping, a novel flow visualization echocardiographic technology, is increasing in popularity. Energy loss reference values for children have been established using vector flow mapping, but those for adults have not yet been provided. We aimed to establish reference values in healthy adults for energy loss, kinetic energy in the left ventricular outflow tract, and the energetic performance index (defined as the ratio of kinetic energy to energy loss over one cardiac cycle). METHODS: Transthoracic echocardiography was performed in fifty healthy volunteers, and the stored images were analyzed to calculate energy loss, kinetic energy, and energetic performance index and obtain ranges of reference values for these. RESULTS: Mean energy loss over one cardiac cycle ranged from 10.1 to 59.1 mW/m (mean ± SD, 27.53 ± 13.46 mW/m), with a reference range of 10.32 ~ 58.63 mW/m. Mean systolic energy loss ranged from 8.5 to 80.1 (23.52 ± 14.53) mW/m, with a reference range of 8.86 ~ 77.30 mW/m. Mean diastolic energy loss ranged from 7.9 to 86 (30.41 ± 16.93) mW/m, with a reference range of 8.31 ~ 80.36 mW/m. Mean kinetic energy in the left ventricular outflow tract over one cardiac cycle ranged from 200 to 851.6 (449.74 ± 177.51) mW/m with a reference range of 203.16 ~ 833.15 mW/m. The energetic performance index ranged from 5.3 to 37.6 (18.48 ± 7.74), with a reference range of 5.80 ~ 36.67. CONCLUSIONS: Energy loss, kinetic energy, and energetic performance index reference values were defined using vector flow mapping. These reference values enable the assessment of various cardiac conditions in any clinical situation.

Difficult tracheal intubation and post-extubation airway stenosis in an 11-month-old patient with unrecognized subglottic stenosis: a case report.

BACKGROUND: Subglottic stenosis can lead to life-threatening difficult tracheal intubation during general anesthesia. We report a case of difficult tracheal intubation in an 11-month-old female who had unrecognized subglottic stenosis. CASE PRESENTATION: The patient was scheduled for elective correction of a right accessory auricle. She was suspected of having first and second branchial arch syndrome. Preoperative physical examination was normal. Anesthesia was induced uneventfully using sevoflurane. It was not possible to pass size 4.0, 3.5, or 3.0 cuffed endotracheal tubes due to an advanced subglottic lesion. Subsequent successful intubation was achieved using a 3.0 uncuffed tube. Stridor was audible after extubation, and the patient required several days' treatment with dexamethasone to address respiratory distress. CONCLUSIONS: We encountered unrecognized subglottic stenosis that led to difficult tracheal intubation and post-extubation airway stenosis.

Effective evaluation of arterial pulse waveform analysis by two-dimensional stroke volume variation-stroke volume index plots.

Arterial pulse waveform analysis (APWA) with a semi-invasive cardiac output monitoring device is popular in perioperative hemodynamic and fluid management. However, in APWA, evaluation of hemodynamic data is not well discussed. In this study, we analyzed how we visually interpret hemodynamic data, including stroke volume variation (SVV) and stroke volume (SV) derived from APWA. We performed arithmetic estimation of the SVV-SV relationship and applied measured values to this estimation. We then collected measured values in six anesthesia cases, including three liver transplantations and three other types of surgeries, to apply them to this SVV-SVI (stroke volume variation index) plot. Arithmetic analysis showed that the relationship between SVV and SV can be drawn as hyperbolic curves. Plotting SVV-SV values in the semi-logarithmic scale showed linear correlations, and the slopes of the linear regression lines theoretically represented average mean cardiac contractility. In clinical measurements in APWA, plotting SVV and SVI values in the linear scale and the semi-logarithmic scale showed the correlations represented by hyperbolic curves and linear regression lines. The plots approximately shifted on the rectangular hyperbolic curves, depending on blood loss and blood transfusion. Arithmetic estimation is close to real measurement of the SVV-SV interaction in hyperbolic curves. In APWA, using SVV as an index of preload and the cardiac index or SVI derived from arterial pressure-based cardiac output as an index of cardiac function, is likely to be appropriate for categorizing hemodynamic stages as a substitute for Forrester subsets.

Pseudomonas aeruginosa Type III Secretory Toxin ExoU and Its Predicted Homologs.

Pseudomonas aeruginosa ExoU, a type III secretory toxin and major virulence factor with patatin-like phospholipase activity, is responsible for acute lung injury and sepsis in immunocompromised patients. Through use of a recently updated bacterial genome database, protein sequences predicted to be homologous to Ps. aeruginosa ExoU were identified in 17 other Pseudomonas species (Ps. fluorescens, Ps. lundensis, Ps. weihenstephanensis, Ps. marginalis, Ps. rhodesiae, Ps. synxantha, Ps. libanensis, Ps. extremaustralis, Ps. veronii, Ps. simiae, Ps. trivialis, Ps. tolaasii, Ps. orientalis, Ps. taetrolens, Ps. syringae, Ps. viridiflava, and Ps. cannabina) and 8 Gram-negative bacteria from three other genera (Photorhabdus, Aeromonas, and Paludibacterium). In the alignment of the predicted primary amino acid sequences used for the phylogenetic analyses, both highly conserved and nonconserved parts of the toxin were discovered among the various species. Further comparative studies of the predicted ExoU homologs should provide us with more detailed information about the unique characteristics of the Ps. aeruginosa ExoU toxin.

The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causing Pseudomonas aeruginosa.

The PcrV cap structure of the type III secretory apparatus of Pseudomonas aeruginosa is a vaccine target. Human immunoglobulin G (IgG) molecules extracted from sera containing high or low anti-PcrV titers were tested for their effects against P. aeruginosa pneumonia in a mouse model. Among 198 volunteers, we selected the top 10 high anti-PcrV titer sera and the bottom 10 low anti-PcrV titer sera and extracted the IgG fraction from each serum sample. First, we examined the effects of the IgG against virulent P. aeruginosa. A lethal dose of P. aeruginosa premixed with saline, low titer human IgG, high titer human IgG, or rabbit-derived polyclonal anti-PcrV IgG was intratracheally administered into the lungs of mice, and their survival and lung inflammation were evaluated for 24 h. The high anti-PcrV titer human IgG had a prophylactic effect. Next, the prophylactic effects of intravenous administration of extracted and pooled high or low anti-PcrV titer human IgG were examined. Here, prophylactic intravenous administration of pooled high anti-PcrV titer human IgG, which showed binding capacity to P. aeruginosa PcrV, was more effective than the administration of its low titer pooled equivalent, and the measured physiological and inflammatory parameters correlated with the anti-PcrV titer levels. This result indirectly implies that high anti-PcrV titers in blood can help to protect against virulent P. aeruginosa infections. In addition, the IgG fractions from such high titer sera have potential to be a source of specific intravenous immunoglobulin products for passive vaccination against virulent P. aeruginosa infections.

Cellular Stress Responses and Monitored Cellular Activities.

To survive, organisms require mechanisms that enable them to sense changes in the outside environment, introduce necessary responses, and resist unfavorable distortion. Consequently, through evolutionary adaptation, cells have become equipped with the apparatus required to monitor their fundamental intracellular processes and the mechanisms needed to try to offset malfunction without receiving any direct signals from the outside environment. It has been shown recently that eukaryotic cells are equipped with a special mechanism that monitors their fundamental cellular functions and that some pathogenic proteobacteria can override this monitoring mechanism to cause harm. The monitored cellular activities involved in the stressed intracellular response have been researched extensively in Caenorhabditis elegans, where discovery of an association between key mitochondrial activities and innate immune responses was named "cellular associated detoxification and defenses (cSADD)." This cellular surveillance pathway (cSADD) oversees core cellular activities such as mitochondrial respiration and protein transport into mitochondria, detects xenobiotics and invading pathogens, and activates the endocrine pathways controlling behavior, detoxification, and immunity. The cSADD pathway is probably associated with cellular responses to stress in human inflammatory diseases. In the critical care field, the pathogenesis of lethal inflammatory syndromes (e.g., respiratory distress syndromes and sepsis) involves the disturbance of mitochondrial respiration leading to cell death. Up-to-date knowledge about monitored cellular activities and cSADD, especially focusing on mitochondrial involvement, can probably help fill a knowledge gap regarding the pathogenesis of lethal inflammatory syndromes in the critical care field.

[Helicopter transportation of a sedated, mechanically ventilated patient with cervical cord injury].

We report helicopter transportation of a sedated, mechanically ventilated patient with cervical cord injury. A 20-year-old male sustained traumatic injury to the cervical spinal cord during extracurricular activities in a college. On arrival at the hospital, a halo vest was placed on the patient and tracheostomy was performed. On the 38th hospital day, he was transported a distance of 520km by helicopter to a specialized hospital in Fukuoka for medical repatriation. Cabin space was narrow. Since power supply and carrying capacity were limited, battery-driven and portable medical devices were used. In consideration for patient's psychological stress, he was sedated with propofol. RSS (Ramsay sedation scale) scores were recorded to evaluate whether the patient was adequately sedated during helicopter transportation. Prior to transport, we rehearsed the sedation using bispectral index monitoring (BIS) in the hospital to further ensure the patient's safety during the transport.