The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

The Effects of Busulfan on Xenogeneic Transplantation of Human Peripheral Blood Mononuclear Cells in Recipient Mice.

BACKGROUND: Humanized mouse models with engraftment of human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) are effective tools for the study of human immunity. Busulfan has been used as a substitute for irradiation in human hematopoietic stem cell (HSC) transplantation models, but it has not been tested in human peripheral blood mononuclear cell (PBMC) transplantation models. METHODS: This study evaluated PBMC engraftment using cytometry and enzyme-linked immunosorbent assay (ELISA) in female NOD.CB17/Prkdcscid/JKrb/ IL2 receptor γ-/- (NIG) mice treated with busulfan. RESULTS: In this model, the percentage of human CD3+ T cell engraftment in the blood was 28.2%, with dominant infiltration of CD8+ cells in the spleen 3 weeks post PBMC transplantation. Production of human cytokines, including Interleukin (IL)-12p70, IL-4, IL-5, IFN-γ, IL-6, IL-8, IL-22, Tumor Necrosis Factor alpha, and IL-10, was determined in mice treated with busulfan. CONCLUSIONS: Our findings demonstrate that busulfan treatment is a beneficial alternative for simple and efficient PBMC engraftment in a rodent model, possibly helping to evaluate human immunity in preclinical studies.

Examining the mechanics of rope bending over a three-dimensional edge in ascending robots.

This paper presents the analysis of ropes' bending on three-dimension edges by ascending robots. A rope ascending robot (RAR) is a type of exterior wall-working robot that utilizes a synthetic rope to traverse the outer surface of a building. Rope-based façade cleaning robots demonstrate effective performance in well-structured buildings. However, in unstructured buildings, the rope used by these robots may become entangled or caught on various structures, presenting a significant challenge for their operation. If the rope becomes caught on a structure, the robot will be unable to move to its intended position. In more severe cases, the rope may become damaged, leading to potential failure or even a fall of the robot. Therefore, solving this problem is crucial for safe and efficient robot operation. Consequently, this study defines the issue of the rope becoming caught on a structure as a rope-locking problem and analyzes it by categorizing it based on the dimensions of contact between the rope and the edge. To address the varying tension experienced in different areas, the rope was divided into micro units and subjected to a three-dimensional analysis to resolve the rope-locking problem. Additionally, the analysis was verified by experiments.

LC-MS-Based Direct Quantification of MicroRNAs in Rat Blood.

MicroRNA (miRNA) has recently garnered significant research attention, owing to its potential as a diagnostic biomarker and therapeutic target. Liquid chromatography-mass spectrometry (LC-MS) offers accurate quantification, multiplexing capacity, and high compatibility with various matrices. These advantages establish it as a preferred technique for detecting miRNA in biological samples. In this study, we presented an LC-MS method for directly quantifying seven miRNAs (rno-miR-150, 146a, 21, 155, 223, 181a, and 125a) associated with immune and inflammatory responses in rat whole blood. To ensure miRNA stability in the samples and efficiently purify target analytes, we compared Trizol- and proteinase K-based extraction methods, and the Trizol extraction proved to be superior in terms of analytical sensitivity and convenience. Chromatographic separation was carried out using an oligonucleotide C18 column with a mobile phase composed of N-butyldimethylamine, 1,1,1,3,3,3-hexafluoro-2-propanol, and methanol. For MS detection, we performed high-resolution full scan analysis using an orbitrap mass analyzer with negative electrospray ionization. The established method was validated by assessing its selectivity, linearity, limit of quantification, accuracy, precision, recovery, matrix effect, carry-over, and stability. The proposed assay was then applied to simultaneously monitor target miRNAs in lipopolysaccharide-treated rats. Although potentially less sensitive than conventional methods, such as qPCR and microarray, this direct-detection-based LC-MS method can accurately and precisely quantify miRNA. Given these promising results, this method could be effectively deployed in various miRNA-related applications.

In vivo genome editing using 244-cis LNPs and low-dose AAV achieves therapeutic threshold in hemophilia A mice.

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244-cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1, locus as a target to enable a combined rebalancing strategy with hF8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40 weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases.

Development of an In Vitro Model for Inflammation Mediated Renal Toxicity Using 3D Renal Tubules and Co-Cultured Human Immune Cells.

BACKGROUND: The emergence of various infectious diseases and the toxic effects of hyperinflammation by biotherapeutics have highlighted the need for in vitro preclinical models mimicking the human immune system. In vitro models studying the relationship between hyperinflammation and acute renal injury mainly rely on 2D culture systems, which have shown limitations in recapitulating kidney function. Herein, we developed an in vitro kidney toxicity model by co-culturing 3D engineered kidney proximal tubules cells (RPTEC/TERT1) with human peripheral blood mononuclear cells (PBMC). METHODS: RPTEC/TERT1 were sandwich cultured to form 3D renal tubules for 16 days. The tubules were then co-cultured with PBMC using transwell (0.4 µm pores) for 24 h. Hyperinflammation of PBMC was induced during co-culture using polyinosinic-polycytidylic acid (polyI:C) and lipopolysaccharide (LPS) to investigate the effects of the induced hyperinflammation on the renal tubules. RESULTS: Encapsulated RPTEC/TERT1 cells in Matrigel exhibited elevated renal function markers compared to 2D culture. The coexistence of PBMC and polyI:C induced a strong inflammatory response in the kidney cells. This hyperinflammation significantly reduced primary cilia formation and upregulated kidney injury markers along the 3D tubules. Similarly, treating co-cultured PBMC with LPS to induce hyperinflammation resulted in comparable inflammatory responses and potential kidney injury. CONCLUSION: The model demonstrated similar changes in kidney injury markers following polyI:C and LPS treatment, indicating its suitability for detecting immune-associated kidney damage resulting from infections and biopharmaceutical applications.

Preclinical Evaluation of interferon-gamma primed human Wharton's jelly-derived mesenchymal stem cells.

The potential of human mesenchymal stem cells (MSCs) for cell therapy has been investigated in numerous immune-mediated conditions; MSCs are considered one of the most promising cellular therapeutics to treat intractable diseases. Recently, approaches to prime MSCs have been investigated, thereby generating cellular products with enhanced potential for a variety of clinical applications. Interferon-gamma (IFN-γ) priming is a current approach used to increase the therapeutic efficacy of MSCs. In this study, we determined the systemic toxicity, tumorigenicity and biodistribution of IFN-γ-primed Wharton's jelly-derived (WJ)-MSCs in male and female BALB/c-nu/nu mice. There were no deaths or pathologic lesions in the mice treated with 5 × 106 cells/kg IFN-γ-primed MSCs in the repeated dose study. In the tumorigenicity study, one of the subcutaneously treated mice showed bronchioloalveolar adenoma in the lung but tested negative for human-specific anti-mitochondrial antibody, suggesting the spontaneous murine origin of the adenoma. A biodistribution study using real-time quantitative polymerase chain reaction demonstrated the systemic IFN-γ-primed MSC clearance by day 28. Based on the toxicity, biodistribution, and tumorigenicity studies, we concluded that IFN-γ-primed MSCs at 5 × 106 cells/kg do not induce tumor formation and adverse changes.

Amelioration of Brain Damage after Treatment with the Methanolic Extract of Glycyrrhizae Radix et Rhizoma in Mice.

Glycyrrhizae Radix et Rhizoma (GR) is a traditional herbal medicine widely used in Asian countries. GR was the most frequently used medicine among stroke patients in Donguibogam, the most representative book in Korean medicine. In the present study, we investigated the neuroprotective effects of the GR methanolic extract (GRex) on an ischemic stroke mice model. Ischemic stroke was induced by a 90 min transient middle cerebral artery occlusion (MCAO), and GRex was administered to mice with oral gavage after reperfusion of MCA blood flow. The MCAO-induced edema and infarction volume was measured, and behavioral changes were evaluated by a novel object recognition test (NORT). Immunofluorescence stains and Western blotting identified underlying mechanisms of the protective effects of GRex. GRex post-treatment in mice with MCAO showed potent effects in reducing cerebral edema and infarction at 125 mg/kg but no effects when the dosage was much lower or higher than 125 mg/kg. GRex inhibited the decrease of spontaneous motor activity and novel object recognition functions. The neuroprotective effects of GRex on ischemic stroke were due to its regulation of inflammation-related neuronal cells, such as microglia and astrocytes.

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. RESULTS: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). CONCLUSIONS: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

A Nonlethal Full-Thickness Flame Burn Produces a Seroma Beneath the Forming Eschar, Thereby Promoting Pseudomonas aeruginosa Sepsis in Mice.

The World Health Organization estimates ~180,000 deaths occur annually from burn-related injuries. Many victims who survive the initial burn trauma succumb to bacterial infections that lead to sepsis during treatment. Although advancements in burn care continue to improve in high-income countries due to their burn centers and advanced research, low and middle-income countries continue to see high frequencies of burn injuries and burn-related deaths due to secondary infections. Bacterial-derived sepsis is the most life-threatening danger for people that survive burn injuries. Here we provide evidence for the first time that a subeschar seroma forms postburn even in the absence of infection in mice. The seroma fills with a volume estimated at 500 µL of fluid, 25% of the blood supply, free of red blood cells. The seroma fluid supports robust Pseudomonas aeruginosa (PA) growth and contains inflammatory cytokines and chemokines, which recruit immature neutrophils and monocytes to the seroma in the absence of endothelial breakdown. These immune cells fail to contain PA expansion and dissemination. This recruitment of monocytes and immature neutrophils may result in sequestering these critical immune cells away from other tissues during a pivotal time during bacterial dissemination, promoting PA-mediated sepsis.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing.

The present study was undertaken to find novel genes associated with colistin resistance in Klebsiella pneumoniae. Five colistin-resistant mutants were derived from four colistin-susceptible parental K. pneumoniae strains belonging to different clones. Whole-genome sequencing was performed for the nine K. pneumoniae strains to screen altered candidate genes. Expression levels of genes with amino acid alterations in derivative strains were determined using quantitative real-time Polymerase chain reaction (PCR). Colistin susceptibility was examined in a parental strain complemented with altered candidate genes. Overall, 13 genetic alterations were identified in five pairs of isogenic K. pneumoniae strains. Genetic alterations related to KP1_3468, including the insertion of an IS5-like element in an intergenic or coding region and amino acid substitutions, were identified in three separate derivative strains. Amino acid substitutions and deletion of PhoQ were determined in one derivative strain. With inactivation of CrrA and substituted CrrB, amino acid substitutions and deletion were identified in a repressor of galETK operon (KP1_0061) and hypothetical protein (KP1_3620), respectively. Decreased colistin susceptibility was observed in a parental strain complemented with KP1-0061, but not a KP1-3620 gene. This study demonstrated diverse genetic paths to colistin resistance in K. pneumoniae. Our results suggest that a repressor of galETK operon may play an important role in colistin resistance in K. pneumoniae.

The Diversity of Lipopolysaccharide (O) and Capsular Polysaccharide (K) Antigens of Invasive Klebsiella pneumoniae in a Multi-Country Collection.

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645 K. pneumoniae isolates were collected from the blood of patients in 13 countries during 2005-2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and wzi capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of K. pneumoniae strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were gmlABC-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and wzi capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent K. pneumoniae invasive infection.

Progress towards the development of Klebsiella vaccines.

Introduction: Klebsiella pneumoniae (KP) are a leading cause of healthcare-associated infections. The dramatic increase in microbial resistance to third-generation cephalosporin and carbapenem 'front line' antimicrobial agents and the paucity of new antimicrobials have left clinicians with few therapeutic options and resulted in increased morbidity and mortality. Vaccines may reduce the incidence of infections thereby reducing the necessity for antimicrobials and are not subject to antimicrobial resistance mechanisms. Areas covered: We review whole cell, subunit, capsular polysaccharide (CPS), O polysaccharide (OPS) and conjugate vaccines against KP infection, as well as alternative KP vaccine platforms. Expert opinion: Vaccine-induced antibodies to KP CPS have been protective in preclinical studies, but the number of CPS types (>77) makes vaccines against this virulence factor less feasible. Since four OPS serotypes account of ~80% of invasive KP infections and anti-OPS antibodies are also protective in preclinical studies, both OPS-based conjugate and multiple antigen presenting system (MAPS) vaccines are in active development. Vaccines based on other KP virulence factors, such as outer membrane proteins, type 3 fimbriae (MrkA) and siderophores are at earlier stages of development. Novel strategies for the clinical testing of KP vaccines need to be developed.

Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa.

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections.

Effect of hypobaria and hyperoxia during sepsis on survival and energy metabolism.

BACKGROUND: Injured warfighters air evacuated to tertiary medical care facilities are subjected to many stresses that may promote the development of sepsis. In this study, we tested the hypothesis that exposure to "in-flight" hypobaria and/or hyperoxia within 24 hours after onset of intra-abdominal infection in rats accelerates the development and/or severity of sepsis and neurologic injury in survivors. METHODS: Sprague-Dawley rats underwent cecal ligation/puncture (CLP) or sham procedures. Twenty-four hours later, rats were then placed in hypobaric chambers for 6 hours and assigned to normobaric conditions and maintained at either 21% or 100% O2, or under hypobaric conditions (pressure equivalent to an altitude of 8,000 ft) but maintained under either 28% or 100% O2. Two days after CLP or sham, blood samples were obtained for cytokine levels, and mitochondria were isolated from the brain and heart of a subset of animals for analysis of mitochondrial oxygen consumption. Animals were also evaluated for neuromotor impairment before and 15 days postsurgery. RESULTS: Among the 70 rats studied, 16.7% of CLP but none of the sham-treated rats died. All of the CLP but none of the sham rats had evidence of peritonitis at 2 days. Twenty percent (6 of 30) CLP rats undergoing hypobaria versus 12.5% (3 of 24) of CLP rats exposed to normobaria died (p = 0.715) while 12% (3 of 25) of CLP rats exposed to hyperoxia versus 20.7% (6 of 29) of CLP rats exposed to normoxia died (p = 0.48). The ratio of mitochondrial ATP-generating O2 consumption to resting respiration was higher in the CLP plus hypobaria under 100% compared with shams. The only difference in H2O2 production was observed in mitochondria from CLP rats exposed to hyperoxia under normobaric conditions. Composite neurologic scores obtained 15 days postinjury were lower than those at baseline for shams. CONCLUSION: We conclude that neither "in-flight" hyperoxia nor hypobaria exacerbate sepsis or neurologic injury.