Long-Term Prognostic Factors in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: A 15-Year Multicenter Retrospective Study.

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a multisystem autoimmune disease with small-vessel involvement. In AAV, microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) are major clinicopathologic variants. In addition, myeloperoxidase (MPO) and proteinase 3 (PR3) are major target antigens. The objective of the study was to explore the predictive factors for long-term survival in AAV patients. Materials and Methods: A multicenter retrospective study was carried out on 407 patients between 2005 and 2020. Clinical parameters were obtained from laboratory tests including the ANCA types, antinuclear antibody (ANA), extractable nuclear antigen (ENA), anti-streptolysin O (ASO), glomerular filtration rate (GFR), and the laboratory examinations for the blood routine, liver function, renal function, and immunity, etc. The data for clinical parameters were collected from electronic medical records (EMRs), and the data for patient survival were acquired through regular follow-up. The association of clinical parameters with overall survival (OS) along with 3-year and 5-year survival rates was analyzed, and the nomogram as a predictive model was established according to the analysis results. Results: In the present study, 336 (82.6%) patients and 46 (11.3%) patients were diagnosed with MPA and GPA, respectively. The mean and median OS for all the patients were 2,285 and 2,290 days, respectively. The 1-year, 3-year, 5-year, and 10-year cumulative survival rates for all the patients were 84.2%, 76.3%, 57.2%, and 32.4%, respectively. Univariate and multivariate survival analyses indicated that the independent prognostic factors included age, pathological categories (MPA, GPA, and other types), serum ANCA types (negative or positive for MPO and/or PR3), ANA, ASO, GFR, lymphocyte, neutrophil-to-lymphocyte ratio (NLR), and C-reactive protein (CRP), and these clinical parameters except for ASO were used to construct a nomogram. The nomogram for 3-year and 5-year survival rates had a C-index of 0.721 (95% CI 0.676-0.766). The calibration curves showed that the predicted values of the nomogram for 3-year and 5-year survival rates were generally consistent with practical observed values, and decision curve analysis (DCA) further demonstrated the practicability and accuracy of the predictive model. Conclusion: Laboratory tests at diagnosis have great significance in the prediction of long-term survival in AAV patients.

[Analysis of blood components of Yougui Yin in normal rats and rats with kidney deficiency caused by adenine based on UPLC-MS technology].

Based on the serum medicinal method, this study aims to investigate the migrating components of Yougui Yin in the blood after intragastric administration, and to provide reference for the basic research of its pharmacodynamics. The kidney deficiency rat model was replicated by adenine method. Normal rats and model rats were administered orally for a single gavage of Yougui Yin. The components in blood were rapidly analyzed and identified by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) and multiple reaction monitoring(MRM), and the migrating components in blood of Yougui Yin were explored by multivariate statistical analysis. The results showed that there were 42 characteristic peaks in the plasma of normal rats by UPLC-Q-TOF-MS technology and 13 chemical components were identified, including 6 alkaloids, 2 flavonoids, 2 triterpenoid saponins, 1 iridoid, 1 phenylpropanoid and 1 monoterpenoid. There were 22 characteristic peaks in the plasma of kidney-deficiency rats, and 12 chemical components were identified, including 2 iridoids, 6 alkaloids, 2 flavonoids, 1 monoterpenoid and 1 triterpenoid saponin. Verbascoside, isoacteoside, acteoside, pinoresinoldiglucoside, loganin and morroniside were identified by MRM both in the plasma of normal rats and kidney-deficiency rats. Compared with 85 monomer components in Yougui Yin, 17 common prototype components were found by UPLC-MS in the plasma of normal rats and kidney deficiency rats, including verbascoside, isoacteoside, acteoside, rehmapicrogenin derived from Rehmanniae Radix Praeparata, pinoresinol diglucoside and geniposidic acid from Eucommiea Cortex, loganin and morroniside derived from Corni Fructus, mesaconine, benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, mesaconitine, aconitine derived from Aconiti Lateralis Radix Praeparata, liquiritin, isoliquiritin and glycyrrhizic acid derived from Glycyrrhizae Radix et Rhizoma. Thirty-one metabolites of medicinal ingredients not found in the plasma of adenine-induced kidney deficiency rats were also detected in the plasma of normal rats. Twelve metabolites of medicinal materials not found in the plasma of normal rats were detected in the plasma of kidney deficiency rats. The results of the study provide reference for explaining the material basis and mechanism of Yougui Yin in the treatment of kidney deficiency.

RGD-binding integrins mediated phagocytosis involved in the entry of Edwardsiella tarda into mudskipper MO/MФ.

The versatile fish pathogen Edwardsiella tarda is an intracellular pathogen with the ability to invade and replicate in host phagocytes. However, the mechanism mediating the uptake of E. tarda in fish monocytes/macrophages (MO/MΦ) is not yet understood. Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E. tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process. In the present study, a total of 1185 up-regulated and 885 down-regulated differentially expressed genes (DEGs) were identified using RNA-seq. Enrichment and pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in pathogen entry. The progress of phagosome formation was observed by transmission electron microscopy. Eight conserved integrin (ITG) subunit genes, belonging to the phagocytic receptors, were found in the transcriptomic sequence data. Additionally, quantitative real-time PCR showed that the mRNA expressions of most ITG subunit genes were related to the different infection times of E. tarda and the different bacterial pathogens. Further assays demonstrated that phagocytosis of FITC-labeled E. tarda by mudskipper MO/MФ was significantly reduced by the tetrapeptide Asp-Gly-Arg-Ser (RGDS). In summary, phagocytosis is one of the entry pathways into mudskipper MO/MΦ, and RGD-binding ITGs are involved in the phagosome formation process.

Ayu (Plecoglossus altivelis) CD46 isoforms protect the cells from autologous complement attack.

CD46 is an important immune regulatory receptor with multiple functions. However, studies on the function of teleost CD46, especially the different CD46 isoforms are limited. In this study, we identified three membrane cofactor protein (MCP, CD46) gene isoforms from ayu (Plecoglossus altivelis) and tentatively named as PaCD46 isoforms. PaCD46 isoforms were generated by alternative splicing and all consisted of four conserved short consensus repeats (SCRs), and the variable serine-threonine-proline-rich domain, transmembrane hydrophobic domain, and cytoplasmic tail. Phylogenetic analysis showed that the isoforms clustered together with other fish CD46 and then with higher animal CD46. Western blotting analysis of peripheral blood mononuclear cells (PBMC) revealed three bands, all of which had much larger molecular weights than the theoretical values of the three PaCD46 isoforms. Moreover, three PaCD46 isoforms were individually expressed on HEK293 cells, and Western blotting showed the similar band profile to that of PBMC. The recombinant extracellular domain of the PaCD46 isoforms, obtained by expression in Pichia pastoris, significantly reduced hemolysis activity of ayu sera. Furthermore, each of the three PaCD46 isoforms respectively protected the HEK293 cells expressing the isoform. The isoforms were also identified for their protection of autologous PBMC from complement activation. These results provided the first evidence that PaCD46 isoforms may be complement regulatory proteins to prevent complement-induced damage to self-tissue.

A soluble FcγR homolog inhibits IgM antibody production in ayu spleen cells.

Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been identified in fish, little is known about the role of such proteins in fish immunity. Here, we cloned a cDNA sequence encoding a soluble Fc receptor for an immunoglobulin G (FcγR) homolog from ayu (Plecoglossus altivelis) (PaFcγRl). The predicted protein was composed of two immunoglobulin C2-like domains but lacked a transmembrane segment and a cytoplasmic tail. The PaFcγRl transcripts were distributed at low levels in all tested tissues, but significantly increased after Vibrio anguillarum infection. The PaFcγRl protein was expressed in the head kidney, trunk kidney, and neutrophils. Recombinant PaFcγRl (rPaFcγRl) was secreted when transfected into mammalian cells and the native protein was also detected in serum upon infection. rPaFcγRl was also demonstrated to bind to ayu IgM, as assessed by cell transfection. Suppressive activity of the recombinant mature protein of PaFcγRl (rPaFcγRlm) on in vitro anti-sheep red blood cell (SRBC) responses was detected by a modified hemolytic plaque forming cell assay. In conclusion, our study revealed that PaFcγRl is closely involved in the negative regulation of IgM production in the ayu spleen.

Characterization and immunologic functions of the macrophage migration inhibitory factor from Japanese sea bass, Lateolabrax japonicus.

Macrophage migration inhibitory factor (MIF) is a cytokine playing critical roles in inflammatory and immune responses. However, its functions have not been well studied in fish. In this study, we identified a MIF molecule from Japanese sea bass (Lateolabrax japonicus; LjMIF). Multiple sequence alignment showed that LjMIF has the typical structural features of MIFs. Phylogenetic tree analysis revealed that LjMIF is most closely related to the yellowfin tuna (Thunnus albacares), large yellow croaker (Larimichthys crocea), and red drum (Sciaenops ocellatus) homologs. Constitutive mRNA expression of LjMIF was detected in all tested tissues, with the highest level in the liver. Upon Vibro harveyi infection, LjMIF transcripts were altered in the tested tissues, including the liver, spleen, and head kidney. Subsequently, we prepared recombinant LjMIF (rLjMIF) and the corresponding antibody (anti-LjMIF). The in vitro study showed that rLjMIF inhibited the trafficking of Japanese sea bass monocytes/macrophages (MO/MΦ) and lymphocytes, but not of neutrophils, while anti-LjMIF had the opposite effect. rLjMIF also enhanced phagocytosis and intracellular killing of V. harveyi by MO/MΦ, while anti-LjMIF only inhibited phagocytosis by MO/MΦ. The in vivo study showed that rLjMIF aggravated the course of V. harveyi infection in Japanese sea bass, but anti-LjMIF increased the survival rate of the fish and decreased the bacterial burden. In conclusion, our observation revealed that LjMIF is closely involved in the immune responses of Japanese sea bass for combating V. harveyi infection.

Ayu C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/macrophages.

The short-chain pentraxins (PTXs), including C-reactive protein (CRP) and serum amyloid P (SAP), are soluble pattern recognition molecules (PRMs) that exhibit calcium-dependent binding to bacterial surface molecules. They opsonize pathogens or other particles by phagocytic clearance. However, the detailed functions of short-chain PTXs in teleosts remained unclear. In this study, we identified a short-chain PTX gene from ayu, Plecoglossus altivelis, and tentatively named as PaCRP/SAP. Sequence analysis revealed that PaCRP/SAP has typical characteristics of fish CRP/SAP and is mostly closely related to rainbow smelt (Osmerus mordax) SAP. PaCRP/SAP transcripts were detected in all tested tissues, with the highest level in the liver, and its expression significantly upregulated following Vibrio anguillarum infection. The active recombinant mature PaCRP/SAP (rPaCRP/SAPm) agglutinated Gram-negative bacteria (Escherichia coli, V. anguillarum, Aeromonas hydrophila, and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) in a calcium-dependent manner in vitro, and it correspondingly bound peptidoglycan and lipopolysaccharide in a dose-dependent manner. The binding of rPaCRP/SAPm to E. coli and S. aureus resulted in a clear inhibition of the deposition of ayu complement 3 (PaC3) on the bacteria. Furthermore, rPaCRP/SAPm decreased phagocytosis of rPaCRP/SAPm-bound E. coli and S. aureus cells by ayu monocytes/macrophages (MO/MΦ) in a complement-dependent way. However, rPaCRP/SAPm alone had no significant influence on phagocytosis. These results provided the first evidence that PaCRP/SAP might function in ayu immune responses via agglutinating bacteria and inhibiting complement-mediated opsonophagocytosis by MO/MΦ.

LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells.

Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization.

Morphology and molecular phylogeny of two colepid species from China, Coleps amphacanthus Ehrenberg, 1833 and Levicoleps biwae jejuensis Chen et al., 2016 (Ciliophora, Prostomatida).

Two colepid ciliates, Coleps amphacanthus Ehrenberg, 1833 and Levicoleps biwae jejuensis Chen et al., 2016, were first recorded in China. Their living morphology, infraciliature and small subunit (SSU) rRNA gene sequences were determined using standard methods. The improved diagnosis of Coleps amphacanthus is as follows:cell size about 100×50 µm in vivo, barrel-shaped; 22-28 ciliary rows each composed of about 14-21 monokinetids and two perioral dikinetids; 5-10 caudal cilia; and one terminal contractile vacuole. Levicoleps biwae jejuensis was also investigated, with an improved diagnosis given based on previous and present work. The phylogenetic analyses based on SSU rRNA gene sequences revealed that all Coleps species were grouped together, except for Coleps amphacanthus, which was grouped into a clade of the genus Levicoleps.

Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis).

Recognizing the presence of invading pathogens by pattern recognition receptors (PRRs) is key to mounting an effective innate immune response. Mammalian CD302 is an unconventional C-type lectin like receptor (CTLR) involved in the functional regulation of immune cells. However, the role of CD302 in fish remains unclear. In this study, we characterized a novel CD302 gene from ayu (Plecoglossus altivelis), which was tentatively named PaCD302. The cDNA sequence of PaCD302 is 1893 nucleotides in length, and encodes a polypeptide of 241 amino acids with molecular weight 27.1 kDa and pI 4.69. Sequence comparison and phylogenetic tree analysis showed that PaCD302 is a type I transmembrane CTLR devoid of the known amino acid residues essential for Ca(2+)-dependent sugar binding. PaCD302 mRNA expression was detected in all tissues and cells tested, with the highest level in the liver. Following Vibrio anguillarum infection, PaCD302 mRNA expression was significantly upregulated in all tissues tested. For further functional analysis, we generated a recombinant protein for PaCD302 (rPaCD302) by prokaryotic expression and raised a specific antibody against rPaCD302. Western blot analysis revealed that the native PaCD302 is glycosylated. Refolded rPaCD302 was unable to bind to five monosaccharides (l-fucose, d-galactose, d-glucose, d-mannose and N-acetyl glucosamine) or two other polysaccharides (lipopolysaccharide and peptidoglycan). It was able to bind to three Gram-positive and seven Gram-negative bacteria, but show no bacterial agglutinating activity. PaCD302 function blocking using anti-PaCD302 IgG resulted in inhibition of phagocytosis and bactericidal activity of ayu monocytes/macrophages (MO/MΦ), suggesting that PaCD302 regulates the function of ayu MO/MΦ. In summary, our study demonstrates that PaCD302 may participate in the immune response of ayu against bacterial infection via modulation of MO/MΦ function.

A transmembrane C-type lectin receptor mediates LECT2 effects on head kidney-derived monocytes/macrophages in a teleost, Plecoglossus altivelis.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine involved in many diseases in which immune dysfunction is present. Ayu LECT2 (PaLECT2), which interacts with a C-type lectin receptor (PaCLR), was shown to activate ayu head kidney-derived monocytes/macrophages (MO/MΦ) to improve the outcomes of fish upon bacterial infections. However, it is not known if PaCLR mediates PaLECT2 effects on ayu MO/MΦ. In this study, we determined the role of PaCLR in signal transduction of PaLECT2 on ayu MO/MΦ. We expressed the PaCLR ectodomain in Escherichia coli and produced a refolded recombinant protein (rPaCLR) that was then used to produce the anti-PaCLR IgG (anti-PaCLR) for neutralization. Addition of the refolded PaLECT2 mature peptide (rPaLECT2m) to ayu MO/MΦ cultures, increased cytokine expression, induced chemotaxis, and enhanced phagocytosis and bactericidal activity of these cells were observed. When we added anti-PaCLR to block the ectodomain of PaCLR, these effects were significantly inhibited. Based on our previous works and the data presented here, we conclude that PaCLR mediates the immunomodulatory effects of PaLECT2 on ayu MO/MΦ, thus defining a mechanism by which LECT2 protects fish against pathogens.

Molecular characterization of an IL-1ß gene from the large yellow croaker (Larimichthys crocea) and its effect on fish defense against Vibrio alginolyticus infection.

Interleukin 1ß (IL-1ß), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1ß gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1ß (LcIL-1ß) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1ß transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1ß transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1ß (rLcIL-1ß) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1ß induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1ß plays an important role in the large yellow croaker immune response against V. alginolyticus.

Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages.

C-type lectin receptors (CTLRs) play vital roles in immune responses as pattern-recognition receptors (PRRs). In this study, we identified a novel C-type lectin receptor (PaCTLRC) gene from ayu, Plecoglossus altivelis. Predicted PaCTLRC is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. Sequence comparison and phylogenetic tree analysis showed that PaCTLRC was most closely related to Atlantic salmon (Salmo salar) CLRC, but was significantly different from two other ayu CTLRs, aCLR and PaCD209L. PaCTLRC transcript was detected in all tested tissues and cells, with high levels in the liver; and its expression was significantly altered upon Vibrio anguillarum infection. Refolded recombinant PaCTLRC (rPaCTLRC) agglutinated three types of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and four types of Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, V. anguillarum and Vibrio parahaemolyticus) in a Ca(2+)-dependent manner in vitro, and Gram-positive bacteria were shown to be biologically relevant ligands for PaCTLRC. rPaCTLRC bound to d-mannose, d-galactose, l-fucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS) and peptidoglycan (PGN), exhibiting a relative binding strength to d-mannose and PGN. d-Mannose, l-fucose, GlcNAc, LPS and PGN could inhibit the agglutinating activity of rPaCTLRC, while d-galactose did not functioned. PaCTLRC neutralization using anti-PaCTLRC IgG resulted in the inhibition of phagocytosis by ayu monocytes/macrophages (MO/MΦ) of S. aureus but not of E. coli, and produced a consistently higher survival rate of S. aureus than that of E. coli. d-Mannose, LPS and PGN treatment had no significant influence on the phagocytosis of ayu MO/MΦ. These results suggest that PaCTLRC may serve as a Gram-positive bacteria-preferred PRR which is involved in pathogen recognition and signal transduction in ayu MO/MΦ.

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414µL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.

Characterization of P2X7R and its function in the macrophages of ayu, Plecoglossus altivelis.

P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.

Molecular characterization of an IL-1ß gene from ayu, Plecoglossus altivelis.

IL-1ß plays a crucial role as a prototypical proinflammatory cytokine in immune responses and has been shown to affect macrophage functions. However, the effects of putative IL-1ß homologs on fish macrophages are still less known. Here, we cloned the full-length cDNA sequence of IL-1ß (aIL-1ß) gene from ayu, Plecoglossus altivelis. Phylogenetic analysis indicated that aIL-1ß was closest to that of Atlantic salmon (Salmo salar). Real-time quantitative PCR (RT-qPCR) revealed that aIL-1ß transcript was mainly expressed in spleen, head kidney and gill, and dramatically increased in various tissues after Listonella anguillarum infection. Subsequently, aIL-1ß was prokaryotic expressed and purified to prepare anti-aIL-1ß antibody. After L. anguillarum challenge, the aIL-1ß mRNA and protein levels were significantly up-regulated in ayu monocytes/macrophages. Moreover, aIL-1ß neutralization did not change phagocytic capability, but reduced bacterial killing capability in ayu head kidney-derived monocytes/macrophages. Therefore, aIL-1ß may play an important role in immune response of ayu, especially, contributing to bacterial killing of monocytes/macrophages.

[Yeast-based production, purification and bioactivity assay of rainbow trout LECT2].

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secretory cytokine that functions in many physiological and pathological processes. We used a Pichia pastoris expression system for the recombinant expression of rainbow trout LECT2. The recombinant LECT2 was purified by UNOsphere S Cation exchange and size-exclusion chromatography columns. The obtained target protein was highly pure (>96% homogeneity) and the yield was >120 mg/L of yeast cultures. An in vitro chamber assay revealed that recombinant LECT2 could induce chemotactic responses in rainbow trout head kidney-derived macrophages. Recombinant LECT2 not only enhanced macrophage respiratory burst activity and bactericidal activity, but also changed macrophage gene expression. In summary, we established a rapid and efficient method to prepare active recombinant rainbow trout LECT2 using a yeast expression system and column chromatography. Bioactive recombinant LECT2 is essential for studies on protein functions.

[Effect of parthenolide on serum expressions of interleukin-1beta and tumor necrosis factor-alpha in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the therapeutic effect of parthenolide (PTL) on rabbit knee arthritis (KOA) and its effects on serum expression of interleukin-1beta (IL-1beta) and contents of tumor necrosis factor-alpha (TNF-alpha). METHODS: Eight rabbits were randomly selected from 40 healthy pure-bred New Zealand rabbits as the normal control group. The KOA model was established in the rest 32 rabbits by plaster cast fixation of the right hind limb extension position. After modeling they were randomly divided into 4 groups, i.e., the model control group, the high dose PTL group, the middle dose PTL group, and the low dose PTL group, 8 in each group. Serum contents of IL-1beta and TNF-alpha were detected using enzyme-linked immunosorbent assay. RESULTS: Compared with the model group, IL-1beta and TNF-alpha concentration decreased in the 3 PTL groups (P < 0.01). The decrement was positively correlated with PTL concentrations (IL-1beta: r = 0.55, P < 0.01; TNF-alpha: r = 0.56, P < 0.01). The inhibition reached the peak when the PTL concentration arrived at 20 micromol/L. CONCLUSIONS: PTL could down-regulate the blood IL-1beta and TNF-alpha concentrations of KOA rabbits. Besides, the decrement was positively correlated with the PTL concentration.

Sequencing of the first ayu (Plecoglossus altivelis) macrophage transcriptome and microarray development for investigation the effect of LECT2 on macrophages.

Macrophages play an important role in first-line host defense of innate immune in fishes. However, it is difficult to investigate cellular mechanism of immune response in fish species with little genomic information available. Here we present the first use of RNA-Sequencing to study the macrophage transcriptome of ayu, Plecoglossus altivelis, which is an economically important fish in East Asia. De novo assembly generated 49,808 non-redundant consensus sequences, among which 23,490 transcripts found respective coding sequences. 15,707 transcripts are predicted to be involved in known metabolic or signaling pathways. The sequences were then used to develop a microarray for measurement the effect of recombinant LECT2 on ayu macrophages. LECT2 altered expression of a variety of genes mainly implicated in actin cytoskeleton, pattern recognition receptors and cytokines. Meanwhile, LECT2 enhanced phagocytosis, bacterial killing, and respiratory burst in ayu macrophages, which supported the thought derived from the microarray data that LECT2 activates macrophages. In conclusion, our results contribute to understanding the specific regulation mechanism of LECT2 in macrophage activation, and the combination of transcriptome analysis and microarray assay is a good method for screening a special tissue or cell response to a stimulus or pathogen in non-model fish species.

LECT2 protects mice against bacterial sepsis by activating macrophages via the CD209a receptor.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine and reduced plasma levels were found in patients with sepsis. However, precise functions and mechanisms of LECT2 remain unclear. The aim of the present study was to determine the role of LECT2 in modulating immune responses using mouse sepsis models. We found that LECT2 treatment improved outcome in mice with bacterial sepsis. Macrophages (MΦ), but not polymorphonuclear neutrophils, mediated the beneficial effect of LECT2 on bacterial sepsis. LECT2 treatment could alter gene expression and enhance phagocytosis and bacterial killing of MΦ in vitro. CD209a was identified to specifically interact with LECT2 and mediate LECT2-induced MΦ activation. CD209a-expressing MΦ was further confirmed to mediate the effect of LECT2 on sepsis in vivo. Our data demonstrate that LECT2 improves protective immunity in bacterial sepsis, possibly as a result of enhanced MΦ functions via the CD209a receptor. The modulation of MΦ functions by LECT2 may serve as a novel potential treatment for sepsis.