Modified activities of macrophages' deubiquitinating enzymes after Francisella infection.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Gram-negative bacterial infection causes aggravated innate immune response in sepsis: Studies from clinical samples and cellular models.

Bacterial infection is the most common cause for sepsis. The purpose of this study was to evaluate the impact of different bacterial infection on sepsis based on human samples and cellular experiments. Physiological indexes and prognostic information of 121 sepsis patients were analysed based on whether they had a gram-positive or gram-negative bacterial infection. Moreover, murine RAW264.7 macrophages were treated with lipopolysaccharide (LPS) or peptidoglycan (PG) to simulate infection with gram-negative or gram-positive bacteria in sepsis, respectively. Exosomes derived from the macrophages were extracted for transcriptome sequencing. In patients with sepsis, most gram-positive bacterial infections were Staphylococcus aureus, and gram-negative infections were Escherichia coli. Gram-negative bacterial infection was significantly associated with high neutrophil and interleukin (IL)-6 levels in blood and shorter prothrombin (PT) and activated partial thromboplastin time (APTT). Intriguingly, the survival prognosis of sepsis patients was not affected by the type of bacterial infection, but it was significantly related to fibrinogen. Protein transcriptome sequencing of the macrophage-derived exosomes showed that differentially expressed proteins were significantly enriched in megakaryocyte differentiation, leukocyte and lymphocyte-mediated immunity, and complement and coagulation cascade pathways. The complement and coagulation-related proteins were significantly upregulated after LPS induction, which explained the shortened PT and APTT in gram-negative bacterial sepsis. Bacterial infection did not affect mortality in sepsis but did alter the host response. The immune disorder induced by gram-negative infection was more severe than that produced by gram-positive infection. This study provides references for the rapid identification and molecular research of different bacterial infections in sepsis.

CD14 signaling mediates lung immunopathology and mice mortality induced by Achromobacter xylosoxidans.

OBJECTIVE AND DESIGN: Our research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model. METHODS: C57Bl/6 or CD14-deficient mice were infected intratracheally with non-lethal inoculum of A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. In another set of experiments, C57Bl/6 or CD14-deficient mice were inoculated with lethal inoculum and the survival rate determined. RESULTS: CD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load. The lungs of CD14-deficient mice presented a smaller area of tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and the levels of sCD14 in BALF and serum of C57Bl/6-infected mice. CONCLUSIONS: In summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.

Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity.

Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (Mϕ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), Mϕ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1ß. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. In vivo, MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.

NLRP3 inflammasome signal pathway involves in Vibrio harveyi-induced inflammatory response in murine peritoneal macrophages in vitro.

Vibrio harveyi, an important zoonotic pathogen, can infect wounds and cause inflammatory response. Understanding the inflammatory response pathways could facilitate the exploration of molecular mechanisms for treating V. harveyi infection. NLR family pyrin domain-containing 3 (NLRP3) inflammasome is involved in the interaction between hosts and pathogenic microorganisms and could be sensed by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Nonetheless, the function of NLRP3 inflammasome in V. harveyi infection remains unclear. In the present study, we established a V. harveyi infection model using murine peritoneal macrophages (PMs). Various techniques, including western blot analysis, enzyme-linked immunosorbent assay (ELISA), RT-qPCR, immunofluorescence, and inhibition assays, were used to explore the molecular mechanism of V. harveyi-induced inflammation. The results showed that many inflammatory cytokines participated in V. harveyi infection, with interleukin (IL)-1ß being the most abundant. Pan-caspase inhibitor pretreatment significantly decreased the secretion of IL-1ß in murine PMs. Moreover, the identification of V. harveyi involved a large number of NLR molecules, especially the NLRP3 receptor, and further studies revealed that NLPR3 inflammasome was activated by V. harveyi infection, as evidenced by puncta-like NLRP3 surrounding cell nuclear, ASC specks in the nucleus and cytoplasm, and ASC oligomerization. Inhibition of NLRP3 inflammasome impaired the release of mature IL-1ß in V. harveyi-infected murine PMs. Furthermore, blocking the secretion of mature IL-1ß could markedly decrease the release of other proinflammatory cytokines, including IL-6, IL-12, and tumor necrosis factor-α. Overall, these data indicated that NLRP3 inflammasome was activated in response to V. harveyi infection and enhanced inflammatory response by promoting IL-1ß secretion in murine PMs.

Combined procalcitonin and hemogram parameters contribute to early differential diagnosis of Gram-negative/Gram-positive bloodstream infections.

BACKGROUND: Hemogram parameters and procalcitonin (PCT) play auxiliary roles in the diagnosis and outcome of sepsis. However, it is not clear whether these indicators can quickly distinguish bacterial classification or guide the choice of empirical antibiotics. METHODS: We retrospectively enrolled 381 patients with bloodstream infections (BSI), divided into Gram-positive bloodstream infections (GP-BSI) and Gram-negative bloodstream infections (GN-BSI). Demographic parameters, hemogram parameters, and PCT were recorded and compared between the two groups. RESULTS: The mean platelet volume (MPV), platelet distribution width (PDW), and PCT in the GN-BSI group were significantly higher than those in the GP-BSI group, while the platelet count (PLT), plateletcrit, platelet count-to-white blood cell count ratio (PWR), platelet count-to-neutrophil count ratio (PNR), platelet count-to-PCT ratio (PLT/PCT), and mean platelet volume-to-PCT ratio (MPV/PCT) were significantly lower in the GN-BSI group. Multivariate stepwise logistic regression analysis revealed that the independent predictors of GN-BSI were MPV, PWR, and PCT. The areas under the curve (AUC) for this prediction model was 0.79, with sensitivity =0.75 and specificity =0.71. CONCLUSIONS: There were significant differences in terms of PCT, platelet parameters, and platelet-related index-PCT ratio between GN-BSI and GP-BSI. Combined PCT and hemogram parameters are more conducive to the early differential diagnosis of bacterial classification of BSI.

Evaluation of Alpha-Ketoglutarate Supplementation on the Improvement of Intestinal Antioxidant Capacity and Immune Response in Songpu Mirror Carp (Cyprinus carpio) After Infection With Aeromonas hydrophila.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Molecular characterization and functional analysis of IL-18 in snakehead (Channa argus) during Aeromonas schubertii and Nocardia seriolae infections.

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1ß-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1ß, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.

Differential mRNA and miRNA Profiles Reveal the Potential Roles of Genes and miRNAs Involved in LPS Infection in Chicken Macrophages.

Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria, and triggers an inflammatory response both in vitro and in vivo. Here, we used LPS from Escherichia coli serotype enteritidis to stimulate chicken macrophages (HD11) and conducted the transcriptome analysis using a bioinformatics approach to explore the functions of immune-related genes and miRNAs. In total, 1759 differentially expressed genes (DEGs) and 18 differentially expressed (DE)-miRNAs were detected during LPS infection. At 6 h post infection, 1025 DEGs and 10 miRNAs were up-regulated, and 734 DEGs and 8 DE-miRNAs were down-regulated. Based on both RNA hybrid and miRanda systems, 55 DEGs could be targeted by 14 DE-miRNAs. The target genes were related to the immune response, such as IRF8, STAT3, TRAF7, and other potential candidate genes. The DE-miRNAs miR146a-3p, miR6583-5p, and miR30c-2-3p were investigated further. They were predicted to target 34 genes that may also be candidates for immune-related miRNAs and genes. Our results enhanced our understanding of the pathogenic mechanisms of Gram-negative bacteria in chickens.

Identification and expression analysis of group II C-type lectin domain containing receptors in grass carp Ctenopharyngodon idella.

Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of ß-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish.

ELAVL1a is an immunocompetent protein that protects zebrafish embryos from bacterial infection.

Previous studies have shown that ELAVL1 plays multiple roles, but its overall biological function remains ill-defined. Here we clearly demonstrated that zebrafish ELAVL1a was a lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs/embryos of zebrafish. ELAVL1a acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, as well as bacteria, but also as an effector molecule, capable of inhibiting the growth of Gram-positive and -negative bacteria. Furthermore, we reveal that the C-terminal 62 residues of ELAVL1a positioned at 181-242 were indispensable for ELAVL1a antibacterial activity. Additionally, site-directed mutagenesis revealed that the hydrophobic residues Val192/Ile193, as well as the positively charged residues Arg203/Arg204, were the functional determinants contributing to the antimicrobial activity of rELAVL1a. Importantly, microinjection of rELAVL1a into embryos markedly promoted their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was considerably reduced by co-injection of anti-ELAVL1a antibody or by knockdown with morpholino for elavl1a. Collectively, our results indicate that ELAVL1a is a maternal immune factor that can protect zebrafish embryos from bacterial infection. This work also provides another angle for understanding the biological roles of ELAVL1a.

Achromobacter xylosoxidans Cellular Pathology Is Correlated with Activation of a Type III Secretion System.

Achromobacter xylosoxidans is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. xylosoxidans plays in pathology remains unknown. This knowledge gap is largely due to the lack of model systems available to study the toxic potential of this bacterium. Recently, a phospholipase A2 (PLA2) encoded by a majority of A. xylosoxidans genomes, termed AxoU, was identified. Here, we show that AxoU is a type III secretion system (T3SS) substrate that induces cytotoxicity to mammalian cells. A tissue culture model was developed showing that a subset of A. xylosoxidans isolates from CF patients induce cytotoxicity in macrophages, suggestive of a pathogenic or inflammatory role in the CF lung. In a toxic strain, cytotoxicity is correlated with transcriptional activation of axoU and T3SS genes, demonstrating that this model can be used as a tool to identify and track expression of virulence determinants produced by this poorly understood bacterium.

IRF8 Regulates Gram-Negative Bacteria-Mediated NLRP3 Inflammasome Activation and Cell Death.

Inflammasomes are intracellular signaling complexes that are assembled in response to a variety of pathogenic or physiologic stimuli to initiate inflammatory responses. Ubiquitously present LPS in Gram-negative bacteria induces NLRP3 inflammasome activation that requires caspase-11. We have recently demonstrated that IFN regulatory factor (IRF) 8 was dispensable for caspase-11-mediated NLRP3 inflammasome activation during LPS transfection; however, its role in Gram-negative bacteria-mediated NLRP3 inflammasome activation remains unknown. In this study, we found that IRF8 promotes NLRP3 inflammasome activation in murine bone marrow-derived macrophages (BMDMs) infected with Gram-negative bacteria such as Citrobacter rodentium, Escherichia coli, or Pseudomonas aeruginosa mutant strain ΔpopB Moreover, BMDMs deficient in IRF8 showed substantially reduced caspase-11 activation and gasdermin D cleavage, which are required for NLRP3 inflammasome activation. Mechanistically, IRF8-mediated phosphorylation of IRF3 was required for Ifnb transcription, which in turn triggered the caspase-11-dependent NLRP3 inflammasome activation in the infected BMDMs. Overall, our findings suggest that IRF8 promotes NLRP3 inflammasome activation during infection with Gram-negative bacteria.

Impaired Airway Epithelial Barrier Integrity in Response to Stenotrophomonas maltophilia Proteases, Novel Insights Using Cystic Fibrosis Bronchial Epithelial Cell Secretomics.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

Diagnostic Accuracy of Procalcitonin Compared to C-Reactive Protein and Interleukin 6 in Recognizing Gram-Negative Bloodstream Infection: A Meta-Analytic Study.

OBJECTIVE: Gram-negative bloodstream infections (GNBSIs), especially those caused by antibiotic-resistant species, have become a public health challenge. Procalcitonin (PCT) showed promising potential in early diagnosis of GNBSI; however, little was known about its performance under different clinical settings. We here systematically assessed the diagnostic accuracy of PCT in recognizing GNBSI and made direct comparisons with C-reactive protein (CRP) and interleukin 6 (IL-6). METHODS: PubMed, Embase, ISI Web of Knowledge, and Scopus were searched from inception to March 15th, 2019. Area under the summary receiver operating characteristic curve (AUC), pooled sensitivity, specificity, and diagnostic odds ratio (DOR) were calculated. Hierarchical summary receiver operating characteristic (HSROC) model was used for the investigation of heterogeneity and for comparisons between markers. RESULTS: 25 studies incorporating 50933 suspected BSI episodes were included. Pooled sensitivity and specificity for PCT were 0.71 and 0.76, respectively. The overall AUC was 0.80. The lowest AUCs were found in patients with febrile neutropenia (0.69) and hematological malignancy (0.69). The highest AUC was found in groups using electrochemiluminescence immunoassay (0.87). In direct comparisons, PCT showed better overall performance than CRP with the AUC being 0.85 (95% CI 0.81-0.87) for PCT and 0.78 (95% CI 0.74-0.81) for CRP, but the relative DORs varied with thresholds between PCT and CRP (p < 0.001). No significant difference was found either in threshold (p < 0.001). No significant difference was found either in threshold (p < 0.001). No significant difference was found either in threshold (. CONCLUSIONS: PCT was helpful in recognizing GNBSI, but the test results should be interpreted carefully with knowledge of patients' medical condition and should not serve as the only criterion for GNBSI. Further prospective studies are warranted for comparisons between different clinical settings.

Effects of thymol supplementation on performance, mortality and branchial energetic metabolism in grass carp experimentally infected by Aeromonas hydrophila.

We determined whether thymol supplementation of would minimize the negative effects of Aeromonas hydrophila infection on branchial energy metabolism, weight loss and mortality in grass carp (Ctenopharyngodon idella). We found that the infected fish all died, while 62.5% of those supplemented with 100 mg/kg thymol survived. Cytosolic and mitochondrial creatine kinase (CK) activities, as well as adenylate kinase (AK) and pyruvate kinase (PK) activities were significant lower in gills of A. hydrophila-infected fish than those of the control group, and adenosine triphosphate (ATP) levels were significant lower in the infected group. Finally, branchial reactive oxygen species (ROS) were significant higher in A. hydrophila-infected fish than in the control group. Supplementation with 100 and 300 mg thymol/kg diet prevented inhibition of branchial cytosolic and mitochondrial CK activities caused by infection, and also inhibited the reduction of branchial ATP levels. Supplementation with 100, 200 and 300 mg thymol/kg prevented the inhibition of branchial AK and PK activities induced by aeromonosis. Supplementation of 100 mg thymol/kg prevented weight loss after A. hydrophila infection. These data suggest that supplementation with 100 mg thymol/kg exerts potent bactericidal properties and augments longevity. Supplementation at all concentrations of thymol prevented A. hydrophila-induced branchial bioenergetics; nevertheless, higher concentrations were associated with side-effects.

Effects of dietary yeast hydrolysate on the growth, antioxidant response, immune response and disease resistance of largemouth bass (Micropterus salmoides).

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ±â€¯0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recommended for largemouth bass based on the present results.

Population pharmacokinetics of amikacin in patients with pediatric cystic fibrosis.

INTRODUCTION: Amikacin is commonly used in patients with pediatric cystic fibrosis (CF) for the treatment of pulmonary exacerbations. Amikacin efficacy is related to maximum plasma concentration/minimum inhibitory concentration (Cmax/MIC) ratio >8. Pharmacokinetic data in patients with pediatric CF are scarce. The aim of this study was to develop a population pharmacokinetic (PopPK) model describing amikacin disposition in patients with pediatric CF. METHODS: CF patients under 18 years of age with pulmonary exacerbation who received amikacin were enrolled. Patients received different amikacin regimens (30 mg-1 kg-1 day-1 every 8, 12, or 24 hours) depending on the patient's status and hospital protocols. Amikacin serum levels were obtained for therapeutic drug monitoring. PopPK model was developed using MONOLIX Suite-2018R1 (Lixoft). RESULTS: A total of 39 patients (114 amikacin concentrations) were included in this study. Population estimates for the elimination rate constant (k) and the volume of distribution (V) were 0.541 hours-1 and 0.451 L/kg, respectively. Between-subject and between-occasion variability were 53% and 16.5% for k and 31% and 22% for V, respectively. Bodyweight was a significant covariate associated with V. Based on simulations, almost 70% of the patients receiving 30 mg-1 kg-1 day-1 every 24 hours would achieve a Cmax/MIC ratio >8 which is an appropriate therapeutic goal while no patient in the other two groups (Q8 and Q12) would achieve that objective. CONCLUSIONS: The regimen of 30 mg-1 kg-1 day-1 every 24 hours more adequately fulfilled the therapeutic target for amikacin. Although all our patients had good clinical results and a good adverse-events profile, further studies are necessary to redefine the optimal treatment strategy.

Dietary addition of rutin impairs inflammatory response and protects muscle of silver catfish (Rhamdia quelen) from apoptosis and oxidative stress in Aeromonas hydrophila-induced infection.

This research aimed to assess the influence of dietary addition of rutin on inflammation, apoptosis and antioxidative responses in muscle of silver catfish (Rhamdia quelen) challenged with Aeromonas hydrophila (A. hydrophila). Fish were split into four groups as follows: control, 0.15% rutin, A. hydrophila, 0.15% rutin + A. hydrophila. After 2 weeks of feeding with standard or rutin diets, fish were challenged or not with A. hydrophila for 1 week. Rutin-added diet abrogates A. hydrophila induced-hemorrhage and inflammatory infiltration. It decreases A. hydrophila induced-apoptosis through decreasing the ratio of Bax to Bcl-2 and increasing phospho-Akt to Akt ratio. It diminishes the A. hydrophila induced-rise in nitric oxide and superoxide anion levels and reestablishes superoxide dismutase activity as well. Although such diet is unable to recover the levels of reduced glutathione (GSH), cysteine and glutamate cysteine ligase, which are depleted as a result of A. hydrophila infection, it diminishes the oxidized glutathione (GSSG) content, thus decreasing GSSG to GSH ratio. It increases the levels of cysteine residues of proteins and diminishes those of thiol-protein mixed disulfides, which were changed after A. hydrophila challenge. Finally, it reduces A. hydrophila induced-lipid peroxidation, markedly elevates ascorbic acid and thus reestablishes total antioxidant capacity, whose levels were decreased after A. hydrophila challenge. In conclusion, the dietary addition of rutin at 0.15% impairs A. hydrophila-induced inflammatory response, inhibits A. hydrophila-induced apoptosis and promotes cell survival. It also reduces the A. hydrophila-induced oxidative stress and stimulates the antioxidative responses in muscle of A. hydrophila-infected silver catfish.

Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.