Pneumonia In Mice Research Articles

Inhibition of the lipoxin A4 and resolvin D1 receptor impairs host response to acute lung injury caused by pneumococcal pneumonia in mice.

Resolution of the acute respiratory distress syndrome (ARDS) from pneumonia requires repair of the injured lung endothelium and alveolar epithelium, removal of neutrophils from the distal airspaces of the lung, and clearance of the pathogen. Previous studies have demonstrated the importance of specialized proresolving mediators (SPMs) in the regulation of host responses during inflammation. Although ARDS is commonly caused by Streptococcus pneumoniae, the role of lipoxin A4 (LXA4) and resolvin D1 (RvD1) in pneumococcal pneumonia is not well understood. In the present experimental study, we tested the hypothesis that endogenous SPMs play a role in the resolution of lung injury in a clinically relevant model of bacterial pneumonia. Blockade of formyl peptide receptor 2 (ALX/FPR2), the receptor for LXA4 and RvD1, with the peptide WRW4 resulted in more pulmonary edema, greater protein accumulation in the air spaces, and increased bacteria accumulation in the air spaces and the blood. Inhibition of this receptor was also associated with decreased levels of proinflammatory cytokines. Even in the presence of antibiotic treatment, WRW4 inhibited the resolution of lung injury. In summary, these experiments demonstrated two novel findings: LXA4 and RvD1 contribute to the resolution of lung injury due to pneumococcal pneumonia, and the mechanism of their benefit likely includes augmenting bacterial clearance and reducing pulmonary edema via the restoration of lung alveolar-capillary barrier permeability.

Identification of potential therapeutic antimicrobial peptides against Acinetobacter baumannii in a mouse model of pneumonia

Acinetobacter baumannii-induced nosocomial pneumonia has become a serious clinical problem because of high antibiotic resistance rates. Antimicrobial peptides (AMP) are an ideal alternative strategy due to their broad-spectrum of antimicrobial activity and low incidence of bacterial resistance. However, their application is limited by toxicity and stability in vivo. The present study used a mouse model to directly identify potential AMPs effective for treatment of A. baumannii-induced pneumonia. Fifty-eight AMPs were screened and two identified (SMAP-29 and TP4) to have prophylactic effects which prevented the death of mice with pneumonia. Furthermore, two TP4 derivatives (dN4 and dC4) were found to have therapeutic activity in pneumonia mouse models by peritoneal or intravenous administration. Both dN4 and dC4 also inhibited and/or eliminated A. baumannii biofilms at higher doses. Taken together, these data suggest the AMP derivatives dN4 and dC4 represent a potential treatment strategy for A. baumannii-induced pneumonia.

Complement and Chlamydia psittaci: Non-Myeloid-Derived C3 Predominantly Induces Protective Adaptive Immune Responses in Mouse Lung Infection.

Recent advances in complement research have revolutionized our understanding of its role in immune responses. The immunomodulatory features of complement in infections by intracellular pathogens, e.g., viruses, are attracting increasing attention. Thereby, local production and activation of complement by myeloid-derived cells seem to be crucial. We could recently show that C3, a key player of the complement cascade, is required for effective defense against the intracellular bacterium Chlamydia psittaci. Avian zoonotic strains of this pathogen cause life-threatening pneumonia with systemic spread in humans; closely related non-avian strains are responsible for less severe diseases of domestic animals with economic loss. To clarify how far myeloid- and non-myeloid cell-derived complement contributes to immune response and resulting protection against C. psittaci, adoptive bone marrow transfer experiments focusing on C3 were combined with challenge experiments using a non-avian (BSL 2) strain of this intracellular bacterium. Surprisingly, our data prove that for C. psittaci-induced pneumonia in mice, non-myeloid-derived, circulating/systemic C3 has a leading role in protection, in particular on the development of pathogen-specific T- and B- cell responses. In contrast, myeloid-derived and most likely locally produced C3 plays only a minor, mainly fine-tuning role. The work we present here describes authentic, although less pronounced, antigen directed immune responses.

Huanglian-Houpo drug combination ameliorates H1N1-induced mouse pneumonia via cytokines, antioxidant factors and TLR/MyD88/NF-κB signaling pathways.

Huanglian-Houpo drug combination (HHDC) is a classical traditional Chinese medicine that has been effectively used to treat seasonal colds and flu. However, no systematic studies of the effects of HHDC on H1N1 influenza infection and the associated mechanisms have been reported. The aim of the present study was to determine the anti-H1N1 influenza effects of HHDC and explore the underlying mechanisms. A mouse model of H1N1-induced pneumonia was established and the mice were treated with HHDC (4, 8 and 16 g/kg) for 5 days after viral challenge. The antiviral effects of HHDC and the underlying mechanisms in the mice were investigated and evaluated with respect to inflammation, oxidative stress and Toll-like receptor (TLR)/myeloid differentiation factor (MyD88)/nuclear factor (NF)-κB signaling pathways. HHDC provided significant protection against weight loss and reduced the H1N1 viral load in the lungs. In addition, HHDC significantly decreased the lung index and increased the spleen and thymus indices of the H1N1-infected mice. HHDC also significantly ameliorated the histopathological changes of pneumonia, decreased serum levels of the cytokines interleukin (IL)-6, tumor necrosis factor-α and interferon-γ, and increased the serum level of IL-2. Moreover, HHDC significantly increased the levels of the antioxidant factors superoxide dismutase and glutathione, and reduced the serum level of nitric oxide. Furthermore, the mRNA and protein expression levels of TLR3, TLR7, MyD88, NF-κB p65 and tumor necrosis factor receptor-associated factor 3 in the lung tissues were significantly decreased by HHDC. These findings suggest that HHDC directly inhibited H1N1 infection in vivo and exerted a therapeutic effect on influenza-induced pneumonia in mice by modulating cytokines, antioxidant factors and TLR/MyD88/NF-κB signaling pathways.

Stroke increases the expression of ACE2, the SARS-CoV-2 binding receptor, in murine lungs

BackgroundThe newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused a worldwide pandemic of human respiratory disease. Angiotensin-converting enzyme (ACE) 2 is the key receptor on lung epithelial cells to facilitate initial binding and infection of SARS-CoV-2. The binding to ACE2 is mediated via the spike glycoprotein present on the viral surface. Recent clinical data have demonstrated that patients with previous episodes of brain injuries are a high-risk group for SARS-CoV-2 infection. An explanation for this finding is currently lacking. Sterile tissue injuries including stroke induce the release of several inflammatory mediators that might modulate the expression levels of signaling proteins in distant organs. Whether systemic inflammation following brain injury can specifically modulate ACE2 expression in different vital tissues has not been investigated. MethodsFor the induction of brain stroke, mice were subjected to a surgical procedure for transient interruption of blood flow in the middle cerebral artery for 45 min and sacrificed after 1 and 3 days for analysis of brain, lung, heart, and kidney tissues. Gene expression and protein levels of ACE2, ACE, IL-6 and IL1β were measured by quantitative PCR and Western blot, respectively. The level of soluble ACE2 in plasma and bronchial alveolar lavage (BAL) was measured using an immunoassay. Immune cell populations in lymphoid organs were analyzed by flow cytometry. Post-stroke pneumonia in mice was examined by bacterial cultures from lung homogenates and whole blood. ResultsStrikingly, 1 day after surgery, we observed a substantial increase in the protein levels of ACE2 in the lungs of stroke mice compared to sham-operated mice. However, the protein levels of ACE2 were found unchanged in the heart, kidney, and brain of these animals. In addition, we found increased transcriptional levels of alveolar ACE2 after stroke. The increased expression of ACE2 was significantly associated with the severity of behavioral deficits after stroke. The higher protein levels of alveolar ACE2 persisted until 3 days of stroke. Interestingly, we found reduced levels of soluble ACE2 in plasma but not in BAL in stroke-operated mice compared to sham mice. Furthermore, stroke-induced parenchymal and systemic inflammation was evident with the increased expression of IL-6 and IL-1β. Reduced numbers of T-lymphocytes were present in the blood and spleen as an indicator of sterile tissue injury-induced immunosuppression. ConclusionsWe demonstrate specific augmented alveolar ACE2 levels and inflammation in murine lungs after experimental stroke. These pre-clinical findings suggest that patients with brain injuries may have increased binding affinity to SARS-CoV-2 in their lungs which might explain why stroke is a risk factor for higher susceptibility to develop COVID-19.

Eriodictyol as a Potential Candidate Inhibitor of Sortase A Protects Mice From Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia.

New anti-infective approaches are urgently needed to control multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Sortase A (SrtA) is a membrane-bound cysteine transpeptidase that plays an essential role in the catalysis of covalent anchoring of surface proteins to the cell wall of Staphylococcus aureus (S. aureus). The present study reports identification of a flavonoid, eriodictyol, as a reversible inhibitor of SrtA with an IC50 of 2.229 ± 0.014 μg/mL that can be used as an innovative means to counter both resistance and virulence. The data indicated that eriodictyol inhibited the adhesion of the bacteria to fibrinogen and reduced the formation of biofilms and anchoring of staphylococcal protein A (SpA) on the cell wall. The results of fluorescence quenching experiments demonstrated a strong interaction between eriodictyol and SrtA. Subsequent mechanistic studies revealed that eriodictyol binds to SrtA by interacting with R197 amino acid residue. Importantly, eriodictyol reduced the adhesion-dependent invasion of A549 cells by S. aureus and showed a good therapeutic effect in a model of mouse pneumonia induced by S. aureus. Overall, the results indicated that eriodictyol can attenuate MRSA virulence and prevent the development of resistance by inhibiting SrtA, suggesting that eriodictyol may be a promising lead compound for the control of MRSA infections.

Hypercapnia selectively modulates LPS-induced changes in innate immune and DNA replication-related gene transcription in the macrophage.

Hypercapnia, the elevation of CO2 in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases and is associated with increased risk of death. Recent studies have shown that hypercapnia inhibits expression of select innate immune genes and suppresses host defence against bacterial and viral pneumonia in mice. In the current study, we evaluated the effect of culture under conditions of hypercapnia (20% CO2) versus normocapnia (5% CO2), both with normoxia, on global gene transcription in human THP-1 and mouse RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We found that hypercapnia selectively downregulated transcription of LPS-induced genes associated with innate immunity, antiviral response, type I interferon signalling, cytokine signalling and other inflammatory pathways in both human and mouse macrophages. Simultaneously, hypercapnia increased expression of LPS-downregulated genes associated with mitosis, DNA replication and DNA repair. These CO2-induced changes in macrophage gene expression help explain hypercapnic suppression of antibacterial and antiviral host defence in mice and reveal a mechanism that may underlie, at least in part, the high mortality of patients with severe lung disease and hypercapnia.

Compound Cocktail Inhibits Influenza Viral Pneumonia via Phospholipase Cγ1 Phosphorylation-Related Necroptosis and Partial Autophagy in Natural Killer Cells.

Influenza viral infections are prone to global outbreaks and cause pneumonia in affected populations. High morbidity and mortality caused by pneumonia occur during an influenza pandemic. Antivirals or control of inflammation is the primary means of influenza treatment. A compound cocktail composed of arctiin, daidzein, glycyrrhizic acid, and liquiritin inhibited mouse pneumonia resulting from a PR8 viral infection and caused a weight gain after oral administration. Natural killer cell activating receptors, both Ly49D and Ly49H in the lungs, were increased in the treatment in mice. In H3N2 virus-infected natural killer-92MI cells, the cocktail treatment had different effects on phosphorylation sites of phospholipase Cγ1 (PLCγ1) and killed infected cells through necroptosis or late apoptosis, in which RIP3 was increased and both caspase-3 and phosphorylated-JNK in the cells were downregulated. Acid phosphatase activity in viral-infected natural killer-92MI cells was induced by the compound cocktail treatment, which could be related to the p62 decrease in natural killer-92MI cells. In addition, an autophagic flux induction was observed in alveolar basal epithelial cells (A549). Protein p65, but not phosphorylated-p65, was significantly decreased by the treatment. Our results indicate that the compound cocktail strengthened the phosphorylation of PLCγ1-related necroptosis and partial autophagy in natural killer cells, which could yield an inhibitory effect on viral pneumonia in influenza.

Prospects for the use of oxidized dextran for the prevention of viral pneumonia and pulmonary fibrosis

Study background. The relevance of the study arises from the large number of fibrotic complications in viral interstitial pneumonia in COVID-19. Objective: to evalu-ate the therapeutic efficacy of inhaling 2% oxidised dex-tran solution in an in vivo model simulating interstitial pneumonia in COVID-19. Materials and methods. The studies were performed on ACR mice. Interstitial pneu-monia in mice was induced by intranasal administra-tion of Escherichia coli lipopolysaccharide (LPS). After intranasal administration of LPS to mice, 2% solution of oxidised dextran or control (saline) solution were inhaled via ultrasonic inhaler. One day later, the animals were killed, and standard histological sections of lung tissue were prepared with subsequent morphometric evalua-tion of the results. Results with data. inhalation of a 2% solution of oxidised dextran makes it possible to almost halve all morphological manifestations of LPS-induced pneumonia in terms of morphometric parameters: the numerical density of vessels with signs of hemocircula-tory disorders, the numerical density of thrombosed vessels, the percentage of thrombosed vessels, the volu-metric density of hemorrhages, the volumetric density atelectasis, volumetric density of cellular inflammatory infiltrate. Conclusion. Inhalation of oxidised dextran so-lutions may be a very promising method for the preven-tion of interstitial pneumonia and pulmonary fibrosis in COVID-19.

1273. Efficacy of Cefiderocol against carbapenem-resistant A. baumannii and P. aeruginosa in ventilator-associated pneumonia mouse model

BackgroundCefiderocol (CFDC) is a novel cephalosporin with siderophore structure, characterized by transportation through siderophore receptor on outer membrane of Gram-negative bacteria and structural stability against beta-lactamase. The antimicrobial activity against multidrug resistant bacteria is demonstrated in vitro and in vivo. In this study, we aimed to elucidate the in vivo efficacy of CFDC using ventilator-associated pneumonia (VAP) mouse model.MethodsThe minimum inhibitory concentration (MIC) of CFDC and meropenem (MEPM) against the test Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) isolates were measured by broth microdilution assay. Iron depleted medium was used for CFDC. For VAP mouse models, neutropenia was induced by cyclophosphamide intraperitoneal administration, followed by intubation of sterile tube in the trachea and inoculation of bacterial suspension. PK analysis were performed in infected mice, in order to determine treatment regimens to achieve targeted time above MIC (TAM) of free concentrations in plasma. Treatment was initiated 3 hours post infection and continued up to 120 h for survival analysis. To investigate the bactericidal effect, the mice were sacrificed to count bacterial load in the lung at 48 h and 24 h for VAP-Ab and Pa, respectively.ResultsMICs(mg/L) of CFDC and MEPM against Ab were 0.5 and 128, and Pa were 0.008 and 16, respectively. The treatment regimens to achieve target MIC were shown in Table 1. In order to assess dose dependency of CFDC, required doses to achieve TAM of 70%, 90%, and 100% were calculated. These doses used in the studies were achievable in human for CFDC, but not for MEPM due to high MICs of the test strains. In treatment study for VAP-Ab, bactericidal effect was achieved at TAM > 70% in CFDC groups, as well as TAM 30% in MEPM group. In VAP-Pa, bactericidal effect was observed at TAM > 90% in CFDC groups, as well as TAM 30% in MEPM group.Table 1.Treatment regimen and free TAM against VAP-Ab and Pa Figure 1. Bacterial load in the lungs of VAP-Ab and Pa ConclusionThe efficacy of CFDC against VAP-Ab and Pa were demonstrated in this study. Although 90% free TAM was required for bactericidal effect, CFDC was shown to be effective against carbapenem-resistant Gram-negative pathogens at the recommended clinical dosing regimen.DisclosuresKatsunori Yanagihara, MD, PhD, Shionogi & Co.,Ltd. (Grant/Research Support)

A New Anti‐Immune Evasion Strategy against Methicillin‐Resistant Staphylococcus Aureus (MRSA) Infections: Simulating Complement Immunotherapy Based on Complement‐Mimic Antibiotic Delivery System

AbstractMethicillin‐resistant Staphylococcus aureus (MRSA) can interfere with synergistic host defenses, thus escaping from immune surveillance. Herein, a new immunotherapy strategy‐simulating complement therapy is proposed. Two artificial effectors, Tat cell penetrating peptide and wheat germ agglutinin, are utilized to develop a multifunctional complement‐mimic liposome (CML) simultaneously delivering antibiotics and orchestrating the complement system with macrophages. The CML successfully simulates the functions of the complement system, including formation of a membrane attack complex, mediation of opsonophagocytosis, and activation of phagocytes. At infection sites, CMLs are able to recognize MRSA and disrupt bacterial permeation barriers (cell walls and cell membranes). Meanwhile, CMLs attached to the surface of MRSA are able to activate the phagocytosis and immune responses of macrophages. As a result, CMLs significantly increased the bacterial activity of clarithromycin both in vitro and in macrophages. Moreover, CMLs effectively reduced the MRSA burden in three infection models, including skin abscesses, pneumonia, and bacteremia mouse models. Therefore, the CML provides a novel strategy for overcoming bacterial immune evasion and sheds light on the development of immunotherapies for infectious diseases.

Overexpression of Macrophage-Inducible C-Type Lectin Mincle Aggravates Proinflammatory Responses to Streptococcus pneumoniae with Fatal Outcome in Mice.

Macrophage-inducible C-type lectin (Mincle)-dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1β cytokine release that was not observed in Glc-DAG-stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG-dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

Impaired host response and the presence of Acinetobacter baumannii in the serum microbiome of type-II diabetic patients.

SummaryType II diabetes (T2D) affects over 10% of the US population and is a growing disease worldwide that manifests with numerous comorbidities and defects in inflammation. This dysbiotic host response allows for infection of the host by numerous microorganisms. In the course of T2D disease, individuals can develop chronic infections including foot ulcers and periodontitis, which lead to further complications and opportunistic infections in multiple body sites. In this study, we investigated the serum of healthy subjects and patients with T2D with (T2DP) or without periodontitis for both microbiome signatures in addition to cytokine profiles. Surprisingly, we detected the presence of Acinetobacter baumanii in the serum of 23% individuals with T2D/T2DP tested. In T2DP, IL-1β, TNF-α, MCP-1, IL-6, IL-8, and IFN-γ were significantly elevated in ABC-positive subjects. As an emerging pathogen, A. baumanii infection represents a risk for impaired inflammation and the development of comorbidities in subjects with T2D.

Construction of Functional Renal Targeting Nano Drug Liposome and Its Effect on Lupus Nephritis

The main purpose of this study was to explore treatment effect of hydroxychloroquine-loaded OX7 nanoliposomes in murine systemic lupus erythematosus (SLE) disease model. Modification of OX7 monoclonal antibody conferred hydroxychloroquine-loaded OX7 nanoliposomes targeting to renal mesangial cells. The SLE mice models were treated with functional nano liposome via tail vein injection, and then the therapeutic effects on lupus nephritis and complicated pneumonia were evaluated. Our research showed that chronic graft versus host disease lupus nephritis mice model has similar characteristic features of renal pathological damage with human SLE, and is reliable for related study. The symptoms and incidence of pneumonia in model mice were significantly alleviated and reduced after treatment with functional nano liposomes prepared in this experiment.

Myeloid arginase-1 controls excessive inflammation and modulates T cell responses in Pseudomonas aeruginosa pneumonia

Regulatory properties of macrophages associated with alternative activation serve to limit the exaggerated inflammatory response during pneumonia caused by Pseudomonas aeruginosa infection. Arginase-1 is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. We investigated the role of arginase-1 in the control of inflammatory immune responses to P. aeruginosa pneumonia in mice that exhibit different immunologic phenotypes. C57BL/6 mice with conditional knockout of the arginase-1 (Arg1) gene from myeloid cells (Arg1ΔM) or BALB/c mice treated with small molecule inhibitors of arginase were infected intratracheally with P. aeruginosa. Weight loss, mortality, bacterial clearance, and lung injury were assessed and compared, as were the characterization of immune cell populations over time post-infection. Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory responses compared to littermate control mice. Arg1ΔM mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to littermate controls. Additionally, Arg1ΔM mice recovered from inflammatory lung injury at a significantly slower rate. Conversely, treatment of BALB/c mice with the arginase inhibitor S-(2-boronoethyl)-l-cysteine hydrochloride (BEC) did not change morbidity as defined by weight loss, but mice at day 10 post-infection treated with BEC had gained significantly more weight back than controls. Neutrophil and macrophage infiltration were similar between groups in the lung parenchyma, and neutrophil migration into the airways was reduced by BEC treatment. Differences seem to lie in the impact on T cell subset disposition. Arg1ΔM mice had increased total CD4+ T cell expansion in the lymph nodes, and increased T cell activation, IFNγ production, and IL-17 production in the lymph nodes, lung interstitium, and airways, while treatment with BEC had no impact on T cell activation or IL-17 production, but reduced the number of T cells producing IFNγ in the lungs. Lung injury scores were increased in the Arg1ΔM mice, but no differences were observed in the mice treated with pharmacologic arginase inhibitors. Overall, myeloid arginase production was demonstrated to be essential for control of damaging inflammatory responses associated with P. aeruginosa pneumonia in C57BL/6 mice, in contrast to a protective effect in the Th2-dominant BALB/c mice when arginase activity is globally inhibited.

Mechanisms of Antiviral Activity of Cistus Salviifolius Extract Against Human Respiratory Viruses

Influenza and ARVIs are the most common forms of infectious respiratory diseases in humans. In this regard, the search and development of means for the prevention and treatment of viral infections is a high priority task. The aim of this study was to assess the mechanisms of the antiviral activity of sage-leaved rock-rose extract (Cistus salviifolius) against the causative agents of influenza and ARVIs in humans. In the course of the study, it was shown that C.salviifolius extract inhibits reproduction of influenza viruses A(H1N1), A (H1N1)pdm09, A(H3N2), A(H5N2), A(H7N9) and influenza B virus. The extract showed maximum virus-inhibiting activity at the early stages of the viral cycle (0–2 hours after infection). C.salviifolius extract significantly reduced the hemagglutinating activity of the virus, and at the same time did not affect the fusogenic properties of viral hemagglutinin. Transmission electron microscopy was used to demonstrate that the cistus extract prevents the absorption of influenza virions on the surface of cells in culture. The inhibitory activity of the extract against other human respiratory viruses, parainfluenza virus and adenovirus, was also shown. The protective activity of C.salviifolius extract was demonstrated when applied intranasally during the experiments on a model of influenza pneumonia in mice. The degree of this activity was in inverse proportion to the time window between the application of the extract and the infection of the animals. The virus, pre-incubated with C.salviifolius extract, did not cause death in the animals. The data obtained indicate that C.salviifolius extract serves as an effective and broad-range means of preventing respiratory viral infections in humans.

Rapid Identification of KL49 Acinetobacter baumannii Associated with Clinical Mortality.

ObjectiveWe aimed to establish a tool for rapid identification of KL49 Acinetobacter baumannii.MethodsBased on the capsular polysaccharide (CPS) synthesis genes database, we investigated the distribution of K locus type 49 (KL49) genes in other KL types and established a rapid identification method for KL49. We collected 61 clinical carbapenem-resistant A. baumannii (CRAB) strains, identified KL49 by gtr100 detection, and used whole genome sequencing (WGS) for verification. A mouse pneumonia model was used to confirm the hypervirulence phenotype. We tested the presence of gtr100 gene in 165 CRAB strains from three provinces in China and evaluated the correlation of gtr100 carrying CRAB infection with mortality.ResultsThe gtr100 gene is the CPS synthesis gene found only in KL49. We screened out nine WGS-validated KL49 strains from 61 CRAB clinical strains using polymerase chain reaction (PCR) to detect the gtr100 gene. The survival rates of KL49 strains were significantly lower than nonKL49 strains in a mouse pneumonia model. The survival rates of LAC-4 gtr100 knockout strain decreased significantly. Analysis of phylogenetics showed the worldwide spread of KL49 A. baumannii. Infection of gtr100 carrying CRAB is an independent risk for mortality (OR, 10.76; 95%CI: 3.08–37.55; p<0.001).ConclusionThe hypervirulence phenotype of KL49 CRAB and the association with mortality highlight the urgent need for implementing control measures. The rapid identification assay has the potential to facilitate early medical intervention and worldwide surveillance.

A Potential Role of Adhesion Molecules on Lung Metastasis Enhanced by Local Inflammation.

Local and systemic inflammations are associated with negative long-term outcomes; however, their precise mechanism of action remains unclear. We previously demonstrated that hepatocyte growth factor (HGF)/c-Met signaling contributed to the enhancement of liver metastasis associated with peritonitis model. The aim of this study is to investigate the effect of local inflammation on the development of lung metastasis. NL-17 cells were injected into BALB/c mice via the tail vein to produce a high potential model for lung metastasis. After injection of NL-17 cells, lipopolysaccharide (LPS) and live Pseudomonas aeruginosa, and phosphate-buffered saline were administered intratracheally to induce acute lung injury (ALI) and pneumonia, respectively. In both ALI and pneumonia mice, lung metastasis was significantly promoted compared to control mice. Concentrations of Interleukin-6, tumor necrosis factor-α, and HGF in the bronchoalveolar lavage fluid were significantly higher in ALI and pneumonia mice than in control mice. Neither administration of recombinant mouse HGF nor c-Met knockdown in NL-17 cells influenced the magnitude of lung metastasis. Yet stimulation with LPS increased the expression of α2 integrin, vascular cell-adhesion protein-1, and intercellular adhesion molecule-1 (ICAM-1) in the lung. Invasive activity of NL-17 cells was significantly up-regulated by LPS, but was suppressed by anti-ICAM-1 antibody. While LPS-stimulated NL-17 cells showed significantly promoted lung metastasis, E-selectin expression in the lungs of mice with ALI or pneumonia was significantly enhanced compared with control mice. Up-regulation of adhesion molecules, but not HGF/c-Met signaling, may contribute to the lung metastasis enhanced by local infection/inflammation.

Examining effects of NS1 specific antibodies on sublethal influenza infection and secondary bacterial pneumonia in mice

Influenza is a highly contagious respiratory disease widespread throughout the world that causes disease in humans, birds and many mammalian species. Annually, around 20% of the global human gets sick with influenza so that more than 500,000 people die its various complications. Secondary bacterial pneumonia poses the peak threat during influenza infection, being most frequently caused by S. pneumoniae. Multiple studies in humans confirm the negative impact of influenza virus infection on subsequent outcome of bacterial pneumonia and provides insight into increased morbidity and mortality due to complicated influenza infection. In particular, the last 2009 influenza pandemic caused by H1N1 virus revealed that 25-56% cases of severe disease forms were associated with secondary pneumonia, among which 14-46% of them were fatal. Based on the aforementioned, it is of high priority to investigate a role of influenza virus proteins in developing of pathogen synergism in viral-bacterial pneumonia, particularly influenza virus non-structural protein NS1. The study objective was to examine effects of NS1-specific antibodies on course of influenza infection and secondary bacterial pneumonia in mice. For this, we used an experimental model of sublethal influenza infection followed by secondary Streptococcus pneumoniae bacterial pneumonia. Influenza A/Puerto Rico/8/34 (H1N1) virus and S. pneumoniae No. 3405 strain were used to simulate influenza infection. Rabbit serum containing antibodies against recombinant NS1 protein from A/Puerto Rico/8/34 virus and native rabbit serum (contain no specific antibodies) were used for vaccination. The study was carried out with female BALB/c mice, weighing 20-22 g. Protective activity of animal serum was assessed by using the three criteria: infection-related mortality, life expectancy and body weight change. The data obtained showed that passive transfer of antibodies specific to influenza virus NS1 protein did not lowered viral replication in sublethal murine model of influenza infection. Subsequent secondary bacterial pneumonia induced by S. pneumoniae revealed no protective effect of anti-NS1 protein antibodies assessed by measuring survival rate, lung viral and bacterial titers in treated vs. control mice.

Treatment of severe pneumonia by hinokitiol in a murine antimicrobial-resistant pneumococcal pneumonia model.

Streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. Antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. Hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against S. pneumoniae in vitro regardless of antimicrobial resistance. In this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. Male 8-week-old BALB/c mice were intratracheally infected with S. pneumoniae strains D39 (antimicrobial susceptible) and NU4471 (macrolide resistant). After 1 h, hinokitiol was injected via the tracheal route. Hinokitiol significantly decreased the number of S. pneumoniae in the bronchoalveolar lavage fluid (BALF) and the concentration of pneumococcal DNA in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. In addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the BALF and serum. Repeated hinokitiol injection at 18 h intervals showed downward trend in the number of S. pneumoniae in the BALF and the concentration of S. pneumoniae DNA in the serum with the number of hinokitiol administrations. These findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. Accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia.