Kunitz-type protease inhibitor TFPI2 remodels stemness and immunosuppressive tumor microenvironment in glioblastoma.

Glioblastoma (GBM) tumors consist of multiple cell populations, including self-renewing glioblastoma stem cells (GSCs) and immunosuppressive microglia. Here we identified Kunitz-type protease inhibitor TFPI2 as a critical factor connecting these cell populations and their associated GBM hallmarks of stemness and immunosuppression. TFPI2 promotes GSC self-renewal and tumor growth via activation of the c-Jun N-terminal kinase-signal transducer and activator of transcription (STAT)3 pathway. Secreted TFPI2 interacts with its functional receptor CD51 on microglia to trigger the infiltration and immunosuppressive polarization of microglia through activation of STAT6 signaling. Inhibition of the TFPI2-CD51-STAT6 signaling axis activates T cells and synergizes with anti-PD1 therapy in GBM mouse models. In human GBM, TFPI2 correlates positively with stemness, microglia abundance, immunosuppression and poor prognosis. Our study identifies a function for TFPI2 and supports therapeutic targeting of TFPI2 as an effective strategy for GBM.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Transcriptome analysis of host anti-Aeromonas hydrophila infection revealed the pathogenicity of A. hydrophila to American eels (Anguilla rostrata).

Aeromonas hydrophila is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-A. hydrophila infection remains uncertain. In this study, LD50 of A. hydrophila to American eels was determined and bacterial load in the liver and kidney of eels was assessed post 2.56 doses of LD50 of A. hydrophila infection. The results showed that the LD50 of A. hydrophila to American eels was determined to be 3.9 × 105 cfu/g body weight (7.8 × 106 cfu/fish), and the bacterial load peaked at 36 h post the infection (hpi) in the liver. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes. Additionally, the results of qRT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 10 hub DEGs and 7 encoded proteins play essential role to the anti-A. hydrophila infection in American eels. Pathogenicity of A. hydrophila to American eels and RNA-seq of host anti-A. hydrophila infection were firstly reported in this study, shedding new light on our understanding of the A. hydrophila pathogenesis and the host immune response to the A. hydrophila infection strategies in gene transcript.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

Pathogenicity of Edwardsiella anguillarum to American eels (Anguilla rostrata) and RNA-seq analysis of host immune response to the E. anguillarum infection.

Edwardsiella anguillarum is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-E. anguillarum infection remains uncertain. In this study, LD50 of E. anguillarum to American eels was determined and bacterial load in the liver and kidney of eels was assessed post the LD50 of E. anguillarum infection. The results showed that LD50 of E. anguillarum to American eels was determined to be 2.5 × 105 cfu/g body weight, and the bacterial load peaked at 36 and 72 h post the infection (hpi) in the kidney and liver, respectively. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes, as well as ultrastructural pathology in the kidney were charactered by acute nephritis, showing necrosis of the renal tubular epithelial cells, glomerular capillaries dilate, mitochondria swelling and ribosomes separate from the endoplasmic reticulum. Furthermore, the results of qRT-PCR revealed that 12 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 6 hub DEGs play essential role to the anti-E. anguillarum infection in American eels. Pathogenicity of E. anguillarum to American eels and hub genes related host anti- E. anguillarum infection were firstly reported in this study, shedding new light on our understanding of the E. anguillarum pathogenesis and the host immune response to the E. anguillarum infection strategies in gene transcript.

Inoculation of Freund's adjuvant in European eel (Anguilla anguilla) revealed key KEGG pathways and DEGs of host anti-Edwardsiella anguillarum infection.

Freund's complete (FCA) and incomplete adjuvants (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the nonspecific immune enhancement. In this study, we examined the RNA-seq in the spleen of European eel (Anguilla anguilla) inoculated with FCA and FIA (FCIA group) to elucidate the key KEGG pathways and differential expressed genes (DEGs) in the process of Edwardsiella anguillarum infection and A. anguilla anti-E. anguillarum infection using genome-wide transcriptome. After eels were challenged by E. anguillarum at 28 d post the first inoculation (dpi), compared to the control uninfected eels (Con group), the control infected eels (Con_inf group) showed severe pathological changes in the liver, kidney and spleen, although infected eels post the inoculation of FCIA (FCIA_inf group) also formed slight bleeding. Compared to the FCIA_inf group, there was more than 10 times colony forming unit (cfu) in the Con_inf group per 100 µg spleen, kidney or blood, and the relative percent survival (RPS) of eels was 44.4% in FCIA_inf vs Con_inf. Compared to the Con group, the SOD activity in the FCIA group increased significantly in the liver and spleen. Using high-throughput transcriptomics, DEGs were identified and 29 genes were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). The result of DEGs clustering showed 9 samples in 3 groups of Con, FCIA and FCIA_inf were similar, contrast to distinct differences of 3 samples in the Con_inf group. We found 3795 up and 3548 down regulated DEGs in the compare of FCIA_inf vs Con_inf, of which 5 enriched KEGG pathways of "Lysosome", "Autophagy", "Apoptosis", "C-type lectin receptor signaling" and "Insulin signaling" were ascertained, and 26 of 30 top GO terms in the compare were significantly enriched. Finally, protein-protein interactions between the DEGs of the 5 KEGG pathways and other DEGs were explored using Cytoscape 3.9.1. The compare of FCIA_inf vs Con_inf showed 110 DEGs from the 5 pathways and 718 DEGs from other pathways formed total of 9747° in a network, of which 9 hub DEGs play vital roles in anti-infection or apoptosis. Together, the interaction networks revealed that 9 DEGs involved in the 5 pathways underlies the key process of A. anguilla anti-E. anguillarum infection or host cell apoptosis.

Role of crustacean female sex hormone in regulating immune response in the mud crab, Scylla paramamosain.

Crustacean female sex hormone (CFSH) is responsible for sexual differentiation in crustaceans. The CFSH exhibited an interleukin-17 domain homologous to vertebrate IL-17, a family of inflammatory cytokines that play vital roles in immune defense. However, the immunoregulation of CFSH in crustaceans is a mystery. Therefore, this study aimed to investigate the immune regulatory roles of CFSH and CFSHR in the mud crab Scylla paramamosain. This study's immunofluorescence result revealed that Sp-CFSHR was highly expressed in granulocytes and semi-granulocytes but had moderate expression in hyalinocytes. The expression level of Sp-CFSH transcript in eyestalk ganglia and Sp-CFSHR in hemocytes were significantly up-regulated by the Poly (I:C) stimulation but significantly down-regulated in response to the lipopolysaccharide (LPS) stimulation. In our study, in vitro experiment exhibited that the nuclear transcription factors NF-κB signaling molecules (Sp-Dorsal and Sp-Relish), Sp-STAT, apoptosis-related gene Sp-IAP, and phagocytosis related gene (Sp-Rab5) expressions were significantly increased in hemocytes by recombinant CFSH (rCFSH) in vitro, but the pro-inflammatory cytokine gene (Sp-IL-16) expression was significantly suppressed. Finally, the rCFSH injection significantly up-regulated Sp-Dorsal, Sp-Relish, Sp-IAP, Sp-Caspase, Sp-ALF2, and C-type lectin (Sp-CTL-B) expressions in hemocytes as well as enhanced the bacterial clearance of the mud crab. In conclusion, our results suggested that CFSH may be a counterpart of vertebrate IL-17 in crustaceans that can enhance innate immunity to defense against Vibrionaceae infection via the NF-κB and/or JAK-STAT signaling pathways. This study provides the first evidence that CFSH is involved in the immunoregulation in crustaceans and enriches the insight of neuroendocrine-immune regulatory system, which providing new ideas for disease prevention in the mud crab industry.

A Short Video Classification Framework Based on Cross-Modal Fusion.

The explosive growth of online short videos has brought great challenges to the efficient management of video content classification, retrieval, and recommendation. Video features for video management can be extracted from video image frames by various algorithms, and they have been proven to be effective in the video classification of sensor systems. However, frame-by-frame processing of video image frames not only requires huge computing power, but also classification algorithms based on a single modality of video features cannot meet the accuracy requirements in specific scenarios. In response to these concerns, we introduce a short video categorization architecture centered around cross-modal fusion in visual sensor systems which jointly utilizes video features and text features to classify short videos, avoiding processing a large number of image frames during classification. Firstly, the image space is extended to three-dimensional space-time by a self-attention mechanism, and a series of patches are extracted from a single image frame. Each patch is linearly mapped into the embedding layer of the Timesformer network and augmented with positional information to extract video features. Second, the text features of subtitles are extracted through the bidirectional encoder representation from the Transformers (BERT) pre-training model. Finally, cross-modal fusion is performed based on the extracted video and text features, resulting in improved accuracy for short video classification tasks. The outcomes of our experiments showcase a substantial superiority of our introduced classification framework compared to alternative baseline video classification methodologies. This framework can be applied in sensor systems for potential video classification.

Immunomodulatory role of crustacean cardioactive peptide in the mud crab Scylla paramamosain.

Crustacean cardioactive peptide (CCAP) is a pleiotropic neuropeptide, but its immunomodulatory role is not clear. Herein, the mud crab Scylla paramamosain provides a primitive model to study crosstalk between the neuroendocrine and immune systems. In this study, in situ hybridization showed that Sp-CCAP positive signal localized in multiple cells in the nervous tissue, while its conjugate receptor (Sp-CCAPR) positive signal mainly localized in the semigranular cells of hemocytes. The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide (LPS) stimulation, but the Sp-CCAP mRNA expression level was up-regulated firstly and then down-regulated after the stimulation of polyriboinosinic polyribocytidylic acid [Poly (I:C)]. After the injection of Sp-CCAP synthesis peptide, the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group. Simultaneously, the mRNA expression of phagocytosis related gene (Sp-Rab5), nuclear transcription factor NF-κB homologues (Sp-Relish), C-type lectin (Sp-CTL-B), prophenoloxidase (Sp-proPO), pro-inflammatory cytokines factor (Sp-TNFSF, Sp-IL16) and antimicrobial peptides (Sp-ALF1 and Sp-ALF5) in the hemocytes were also significantly up-regulated at different time points after the injection of Sp-CCAP synthetic peptide, but Sp-TNFSF, Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h. In addition, RNA interference of Sp-CCAP suppressed the phagocytic activity of hemocytes and inhibited the mRNA expression of Sp-Rab5, Sp-Relish, Sp-CTL-B, Sp-TNFSF, Sp-IL16 and Sp-ALF5 in the hemocytes, and ultimately weakened the ability of hemolymph bacteria clearance of mud crab. Taken together, these results revealed that CCAP induced innate immune and increased the anti-infection ability in the mud crab.

Transcriptomic analysis using dual RNA sequencing revealed a Pathogen-Host interaction after Edwardsiella anguillarum infection in European eel (Anguilla anguilla).

Many studies have explored differentially expressed genes (DEGs) between some pathogens and hosts, but no study has focused on the interaction of DEGs between Edwardsiella anguillarum (Ea) and Anguilla anguilla (Aa). In this study, we examined the interactions of DEGs during Ea infection and Aa anti-infection processes by dual RNA sequencing. Total RNA from in vitro and in vivo (Aa liver) Ea culture was extracted. Using high-throughput transcriptomics, significant DEGs that were expressed between Ea cultured in vitro versus in vivo and those in the liver of the infected group versus control group were identified. Protein-protein interactions between the pathogen and host were explored using Cytoscape according to the HPIDB 3.0 interaction transcription database. The results showed that the liver in the infection group presented with severe bleeding and a large number of thrombi in the hepatic vessels. We found 490 upregulated and 398 downregulated DEGs of Ea in vivo versus Ea cultured in vitro, and 2177 upregulated and 970 downregulated genes in the liver of the infected eels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathogen DEGs revealed that the upregulated genes were mainly enriched in migration, colonization, biofilm formation, and significantly enriched in ABC transport and quorum sensing; the downregulated genes were mainly involved in metabolism, information transduction, organelle formation, enzyme catalysis, molecular transport, and binding. GO of the host DEGs showed that metabolic process, catalytic activity, single organism metabolic process, small molecule binding, nucleotide binding, nucleotide phosphate binding, and anion binding were markedly enriched. Finally, we found that 79 Ea and 148 Aa proteins encoded by these DEGs were involved in an interaction network, and some pathogen (DegP, gcvP, infC, carB, rpoC, trpD, sthA, and FhuB) and host proteins (MANBA, STAT1, ETS2, ZEP1, TKT1, NMI and RBPMS) appear to play crucial roles in infection. Thus, determining the interaction networks revealed crucial molecular mechanisms underlying the process of pathogenic infection and host anti-infection.

Erratum: PDLSCs Regulate Angiogenesis of Periodontal Ligaments via VEGF Transferred by Exosomes in Periodontitis: Erratum.

[This corrects the article DOI: 10.7150/ijms.40918.].

Transcriptome of Edwardsiella anguillarum in vivo and in vitro revealed two-component system, ABC transporter and flagellar assembly are three pathways pathogenic to European eel (Anguilla anguilla).

Edwardsiella anguillarum is one of the common bacterial pathogens for the cultivated eels in China. The aim of this study was to reveal the cause of E. anguillarum pathogenic to European eel (Anguilla anguilla) from the perspective of the transcriptome. In this study, we first prepared E. anguillarum cultured in vitro and analysed the whole transcriptome after extracting the total RNA. Then, eels were i.p injected with E. anguillarum, and total RNA were extracted from the liver of European eels 48 h after the infection. After sequencing the transcriptome, we obtained average 1.97 × 108 clean reads cultured in vitro and 1.36 × 105 clean reads located in vivo after annotating all reads into the genome of E. anguillarum. The whole transcriptome showed, compared to the E. anguillarum cultured in vitro, 503 significantly up and 657 significantly down-regulated different expressed genes (DEGs) were observed. KEGG analysis showed that 38 DEGs of Two-Component System, 41 DEGs of ABC transporter, and 10 DEGs flagellar assembly pathways were highly upregulated in E. anguillarum located in vivo. Then, we designed primers to analyse the up-regulated DEGs through qRT-PCR and confirmed some up-regulated DEGs. The results of this study provide important reference for the further study of pathogen-host interaction between E. anguillarum and European eel.

Transcriptome RNA-seq revealed lncRNAs activated by Edwardsiella anguillarum post the immunization of OmpA protecting European eel (Anguilla anguilla) from being infected.

Long noncoding RNAs (lncRNAs) play important roles in various biological activities as vital regulators. However, no study has focused on the lncRNA regulation of Outer membrane protein (OMP) immunization against aquatic bacterial infection. In this study, we examined the genome-wide expression of lncRNAs in the liver of European eel (Anguilla anguilla, Aa) administrated by a recombinant OmpA (rOmpA) from Edwardsiella anguillarum (Ea) to elucidate the functions of lncRNAs in the process of Ea infection and Aa anti-Ea infection using strand specific RNA-seq. Eels were challenged by Ea at 28 d post the immunization (dpi) of OmpA, and the result showed, compared to uninfected livers in the PBS group (Con group), the infected livers in the PBS group (Con_inf group) showed severe bleeding, hepatocyte atrophy and thrombi formed in the hepatic vessels; livers in the OmpA group (OmpA_inf) also formed slight thrombi in the hepatic vessels. The relative percent survival of eels in OmpA_inf vs Con_inf was 78.6%. Using high-throughput transcriptomics, we found 13405 lncRNAs in 3 compares of Con_inf vs Con, OmpA_inf vs Con and OmpA_inf vs Con_inf, of which 111, 129 and 158 DE-lncRNAs were ascertained. GO analysis of the DE-lncRNAs revealed the targeting DEGs were mainly involved in single-organism process, signaling, biological process and response to stimulus in BP, component of membrane in CC and binding in MF; KEGG pathways showed that the targeting DEGs in co-expression and co-location enriched in cell adhesion molecules. Finally, 54 DE-lncRNAs targeting 1675 DEGs were involved in an interaction network of 21692 co-expression and 483 co-location related links, of which 18 DE-lncRNAs appear to play crucial roles in anti-Ea infection. Thus, the interaction networks revealed crucial DE-lncRNAs underlying the process of Ea infection and Aa anti-Ea infection pre and post the immunization of OmpA.

Short neuropeptide F enhances the immune response in the hepatopancreas of mud crab (Scylla paramamosain).

Short neuropeptide F (sNPF), a highly conserved neuropeptide, displays pleiotropic functions on multiple aspects of physiological processes, such as feeding, metabolic stress, locomotion, circadian clock and reproduction. However, to date there has no any report on the possible immunoregulation of sNPF in crustaceans. In the present study, we found that the Sp-sNPF was mainly expressed in the nervous tissue in the mud crab Scylla paramamosain, while the sNPF receptor gene (Sp-sNPF-R) was expressed in a wide variety of tissues, including the hepatopancreas. In situ hybridization further showed that the Sp-sNPF-R positive signal mainly localized in the F-cells of the hepatopancreas. Moreover, the Sp-sNPF-R transcription could be significantly up-regulated after the challenge of bacteria-analog LPS or virus-analog Poly (I:C). Both in vitro and in vivo experiments showed that the synthetic sNPF peptide significantly increased the gene expressions of sNPF-R, nuclear factor-κB (NF-κB) signaling genes and antimicrobial peptides (AMPs) in the hepatopancreas. Simultaneously, the administration of sNPF peptide in vitro also increased the concentration of nitric oxide (NO) and the bacteriostasis of the culture medium of hepatopancreas. These results indicated that sNPF up-regulated hepatopancreas immune responses, which may bring new insight into the neuroendocrine-immune regulatory system in crustacean species, and could potentially provide a new strategy for disease prevention and control for mud crab aquaculture.

Immunization of a novel bivalent outer membrane protein simultaneously resisting Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, European eels (Anguilla anguilla) were immunized by the bivalent expression products of the outer membrane protein (Omp) gene from A. hydrophila (OmpⅡ) and E. anguillarum (OmpA), and the effects of the bivalent protein (rOmpⅡ-A) on the immune function of the European eel were detected. Three hundred eels were divided average into three groups of PBS, adjuvant and rOmp. Eels of three goups were injected intraperitoneal with 0.2 mL of PBS (0.01 mol/L, pH7.4), PBS + F (PBS mixed equal volume of freund's uncomplete adjuvant) or rOmpⅡ-A (1 mg mL-1 rOmpⅡ-A mixed equal volume of freund's uncomplete adjuvant). Four immune-related genes expression, proliferation of whole blood cells, serum and skin mucus antibody titer, superoxide dismutase (SOD) activity and the relative percent of survival (RPS) were studied at different days (or hours) post the immunization. The results showed that the igm, lysC, mhc2 and sod gene in the liver, spleen, kidney and intestine tract were significant increased in the Omp group; On the 28 day post the immunization (dpi), blood cell proliferation was increased in the Omp group, and on the 14, 21, 28 and 42 dpi, antibody titers in serum and mucus of the Omp group were significantly higher than that of the PBS and adjuvant group, regardless of coating with bacteria or Omp antigen. The SOD activity of Omp group increased significantly in liver, kidney, skin mucus and serum from 14 to 42 dpi, especially in serum. Eels chanllenged by A. hydrophila, E. anguillarum and V. vulnificus in the bivalent Omp group showed the RPS were 83.33%, 55.56% and 44.44%, respectively. The results of this study showed that immunization of the bivalent Omp could effectively improve the immune function of European eels, and produced effectively protection to A. hydrophila and E. anguillarum infection. Simultaneously, the bivalent Omp also produced distinct cross-protection to the eels challenged by V. vulnificus.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL-1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.

PDLSCs Regulate Angiogenesis of Periodontal Ligaments via VEGF Transferred by Exosomes in Periodontitis.

Abnormal angiogenesis is one of the significant features in periodontitis leading to progressive inflammation, but angiogenic changes of periodontal ligaments under inflammatory condition were rarely reported. Periodontal ligament stem cells (PDLSCs) were a kind of dental stem cells associated with vascularization. Here we investigated the alteration of angiogenesis of periodontal ligament in periodontitis, and revealed an exosome-mediated pathway to support the effect of PDLSCs on angiogenic improvement. Vascular specific marker CD31 and VEGFA were found to be highly expressed in periodontal ligaments of periodontitis. The VEGFA expression was up-regulated in inflamed PDLSCs compared to control, meanwhile the tube formation of HUVECs was improved when co-cultured with inflamed PDLSCs. Exosomes secretion of PDSLCs was augmented by inflammation, and promoted angiogenesis of HUVECs, whereas blocking secretion of exosomes led to degenerated angiogenesis of HUVECs. Exosome-trasferred VEGFA was proven to be the crucial communicator between PDLSCs and HUVECs. Inflammation inhibited miR-17-5p expression of PDLSCs and relieved its target VEGFA. However, overexpression of miR-17-5p blocked the pro-angiogenic ability of inflamed PDLSCs. In conclusion, the findings indicated that vascularization of periodontal ligaments was enhanced, and inflammatory micro-environment of periodontitis facilitated pro-angiogenesis of PDLSCs through regulating exosome-mediated transfer of VEGFA, which was targeted by miR-17-5p.

Construction expression and immunogenicity of a novel trivalent outer membrane protein (OmpU-A-II) from three bacterial pathogens in Japanese eels (Anguilla japonica).

Vibrio vulnificus, Edwardsiella anguillarum and Aeromonas hydrophila are three common bacterial pathogens in cultivated eels. To protect farming eels from infection by these pathogens, a trivalent outer membrane protein (OMP) containing partial sequences of OmpU from V. vulnificus, OmpA from E. anguillarum and OmpII from A. hydrophila was expressed and purified; then, the OMP was used as a vaccine to immunize Japanese eels (Anguilla japonica). Whole-blood cell proliferation, antibody titres and complement and lysozyme activities were detected at different days post-immunization (dpi), and the relative per cent survival (RPS) was determined after eels were infected with V. vulnificus, E. anguillarum or A. hydrophila at 28 dpi. The results showed that the OMP significantly stimulates the antibody titres. At 14 days after the challenge (i.e. at 28 dpi), the RPS of OMP against V. vulnificus, E. anguillarum and A. hydrophila was 20%, 70% and 11.1%, respectively. The construction, expression and immunogenicity of a trivalent Omp were reported for the first time, and this study will provide a valuable reference for the development of fish multiplex vaccines.

Six-month results of stenting of the femoropopliteal artery and predictive value of interleukin-6: Comparison with high-sensitivity C-reactive protein.

OBJECTIVES: To determine the association of pre- and postinterventional serum levels of interleukin-6 and high-sensitivity C-reactive protein at the six-month evaluation of restenosis after stenting of the femoropopliteal artery. METHODS: Sixty-eight consecutive patients with steno-occlusive femoropopliteal artery disease of Rutherford category III or IV who underwent stent implantation were included. Six-month patency was evaluated with color-coded duplex ultrasound. The association of in-stent restenosis with interleukin-6 and high-sensitivity C-reactive protein levels at baseline, and 24-h postintervention was assessed with a multivariate logistic regression analysis. RESULTS: In-stent restenosis was found in 15 patients (22.1%) within six months. Interleukin-6 and high-sensitivity C-reactive protein levels were significantly increased at 24-h postintervention compared to their preintervention values (p < 0.001 and p = 0.002, respectively). Interleukin-6 values at baseline (odds ratio, 1.11; 95% confidence interval: 1.00, 1.23; p = 0.044) and 24-h postintervention (odds ratio, 1.04; 95% confidence interval: 1.02, 1.06; p < 0.001) were independently associated with six-month in-stent restenosis. Twenty-four-hour postinterventional high-sensitivity C-reactive protein levels were also found to be related to restenosis (odds ratio, 1.15; 95% confidence interval: 1.04, 1.26; p = 0.006), but high-sensitivity C-reactive protein levels at baseline did not show an independent association with in-stent restenosis (odds ratio, 0.57; 95% confidence interval: 0.35, 1.80; p = 0.667). Smoking, diabetes mellitus, and cumulative stent length were other parameters associated with an increased risk for in-stent restenosis. CONCLUSIONS: Femoropopliteal artery angioplasty with stent placement induces an inflammatory response. Interleukin-6 is a powerful independent predictor of intermediate-term outcomes for stenting of the femoropopliteal artery, suggesting that its predictive value may be superior to that of high-sensitivity C-reactive protein.