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The present work provides information concerning the immunostimulatory activity of Ergosan, an algal based product, injected intraperitoneally in the rainbow trout (Oncorhynchus mykiss). Ergosan is composed of 0.002% unspecified plant extract, 1% alginic acid from Laminaria digitata, and 98.998% algal based carrier. Migration of leucocytes into the peritoneal cavity was stimulated at doses > or =1 mg ml(-1). A single dose of 1mg significantly augmented the proportion of neutrophils, degree of phagocytosis, respiratory burst activity and expression of interleukin-1beta (IL-1beta), interleukin-8 (IL-8) and one of the two known isoforms of trout tumour necrosis factor-alpha (TNF2) in peritoneal leucocytes at 1 day post-injection. Humoral immune parameters were less responsive to intraperitoneal Ergosan administration, with complement stimulation only evident in the 1mg treated group at 2 days post-injection. Antiprotease and lysozyme activity were unaffected by Ergosan over a 7-day time period at the doses examined.  相似文献   

4.
The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1-1000ng/mL) for various times. Recombinant IL-10 (10-5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.  相似文献   

5.
To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.  相似文献   

6.
Interleukin (IL)-1beta-encoding regions of chicken, duck, goose, turkey and pigeon were cloned and sequenced. Each IL-1beta-encoding region of chicken, duck, goose and turkey is 804 nucleotides long and encodes IL-1beta protein that is 268 amino acids. Pigeon IL-1beta-encoding region is 810 nucleotides long and encodes IL-1beta protein that is 270 amino acids. Two one-nucleotide and one four-nucleotide insertions of pigeon IL-1beta-encoding region sequence were found, resulting in two amino acid insertions in pigeon IL-1beta. Pairwise sequence analysis showed that the sequence identities of IL-1beta-encoding genes ranged from 77% to 99%, which were also found for IL-1beta protein sequence identities, with an average level of both sequence identities of 89%. Phylogenetic analysis indicated that IL-1beta-encoding regions and the encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from the binding reaction of antiserum against each recombinant IL-1beta (r IL-1beta) protein to homologous or heterologous rIL-1beta, the enhancement levels of K60 mRNA expression in rIL-1beta-treated DF-1 cells or the reduction levels of K60 mRNA expression in DF-1 cells treated with rIL-1beta that was preincubated with homologous or heterologous antiserum showed that all five rIL-1beta were functional active and shared significantly structural and functional homology.  相似文献   

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Chlorinated dioxins, as typified by the most potent isomer, TCDD, are immunosuppressive in mammalian species and can enhance the susceptibility to a number of diseases. In recent years chlorinated dioxins have been detected in fish in many freshwater and marine habitats. Thus far, the effects of these chemicals on the immune responses of fish have not been examined. We studied the influence of TCDD on the defense mechanisms of rainbow trout. Yearling trout were injected intraperitoneally with the vehicle, 0.1, 1.0 or 10 micrograms/kg of TCDD. Interactions with the humoral immune response to sheep red blood cells (SRBC) were assessed by the Jerne plaque assay using head kidney and spleen leukocytes. Serum antibody was measured by complement-mediated lysis of SRBC in a chromium release assay. Effects of TCDD on the cellular immune responses were evaluated by the response of thymic and splenic lymphocytes to Con A and PWM. In addition, the phagocytic activity of peritoneal macrophages was examined in vitro. Trout which received 0.1 or 1.0 micrograms/kg TCDD remained clinically normal, and defense mechanisms were unaltered in these fish. Trout which received 10 micrograms/kg of TCDD became hypophagic and exhibited fin necrosis, ascites and suppression of hematopoiesis. In this treatment group, Con A-induced blastogenesis of thymic and splenic lymphocytes was not significantly changed, however, suppression of the PWM-induced response of splenic lymphocytes occurred. No statistically significant alterations occurred in humoral immune responses, and phagocytic activity of peritoneal macrophages was not decreased. The dose-response curve for various biologic effects of TCDD in the rainbow trout appears different from that in sensitive mouse strains. The 30-day, single-dose, parenteral LD50 for TCDD in the C57BL mouse is 100 micrograms/kg, and TCDD suppresses both cell-mediated and humoral immune responses at 1-2 micrograms/kg in this mouse strain. In the rainbow trout, however, immunosuppression was evident only at doses of TCDD approaching the 80-day, single-dose, parenteral LD50 of 20 micrograms/kg.  相似文献   

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Bacterial DNA and CpG ODN have both been shown to have immunostimulatory effects in mammals, activating APCs and inducing a potent Th1 type immune response. They have also been shown to have a strong adjuvant effect and up-regulate MHC class 2 expression in murine cells, augment human and murine NK cell lytic activity, activate human B cells and induce murine B cell proliferation. However, little work has been carried out with regard to their effects on the piscine immune system. Here it is shown that various CpG ODN induce proliferation of peripheral blood leucocytes, spleen and head kidney cells from rainbow trout although, at the range of concentrations tested CpG ODN 2133 lacked the ability to induce specific antibody production to a protein antigen.  相似文献   

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在SD大鼠成骨细胞(Osteoblast,OB)体外培养体系中添加不同浓度1α,25-二羟维生素D3(0、10^-9、10^-8、10^-7mol/L),作用24、48、72h,测定OB增殖率、碱性磷酸酶(ALP)活性,作用48h流式细胞仪测定0B周期。结果显示,10^-9mol/L 1α,25-二羟维生素D3作用24、48、72h均促进oB增殖(P〈0.05或P〈0.01),抑制ALP活性(P〈0.01);10^-8、10^-7mol/L作用24、48h,OB增殖率与对照组差异不显著(P〉0.05),但24h时ALP活性均明显升高(P〈0.05或P〈0.01),48h则抑制了ALP活性并使OB滞留在G2/M期(P〈0.05或P〈0.01);72h时10^-7mol/L组OB增殖率极显著低于其余各组(P〈0.01),并使ALP活性升高(P〈0.01)。表明低浓度1α,25-二羟维生素D3能促进OB增殖,抑制其分化;高浓度1α,25-二羟维生素D3能抑制OB增殖,促进其分化,并使细胞滞留在G2/M期。  相似文献   

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The effects of administration of the immunomodulator Ergosan, an algal extract containing alginic acid, in juvenile rainbow trout (Oncorhynchus mykiss) exposed to AquaVac vaccination, were tested. Juveniles treated with Ergosan, 95 days after the beginning of first solid feeding and control fish fed solely on commercial diet, were vaccinated by immersion in AquaVac solution. The time-course of the effects of vaccination on liver immunorelated gene modulation and on the tolerance to stress manipulation connected with the vaccination was investigated. Liver and plasma sampling was performed at the following times: T=pre-vaccination, T0=5min, T1=2h, T2=8h, T3=24h, T4=48h and T5=72h post-vaccination. Interleukin-1beta (IL-1beta), interleukin-8 (IL-8), tumor necrosis factor alpha 2 (TNF alpha 2) and heat shock protein 70 (Hsp70) gene expression in trout liver was monitored by real-time PCR using Acidic Ribosomal Phosphoprotein P0 (ARP) as internal standard. The evaluation of the plasma cortisol levels was performed by EIA. In AquaVac-vaccinated fish, both the gene expression of Hsp70 and the plasma cortisol levels during the time-course were significantly (P<0.05) lower in Ergosan-treated fish with respect to control, indicating the positive role of Ergosan on handling stress tolerance. This study also demonstrated the stimulatory properties of Ergosan on cytokine genes expression involved in innate immune response: liver IL-1beta, IL-8 and TNF alpha 2 gene expression was significantly (P<0.05) higher in trout fed on Ergosan compared to control, indicating a positive role of this feed additive in improving the immune responsiveness to AquaVac vaccine.  相似文献   

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This work describes the characterization of trout haematopoietic in vitro long-term cell cultures showing specific viral antigen-dependent cell (ADC) proliferation. The ADC cultures were developed from outbreed trout after surviving viral hemorrhagic septicaemia virus (VHSV) infections or after immunization with purified VHSV. For in vitro long-term proliferation of the ADC cultures, adherent (Ad) cells obtained from autologous trout were pulsed with VHSV recombinant glycoprotein G4 (G4-pulsed Ad cells) and added periodically to the cultures. ADC did not proliferate in cultures obtained from non-infected control trout treated in parallel with G4 or from VHSV survivor/VHSV immunized trout kidney donors treated with non-viral proteins. After months in culture, the ADC acquired an increasingly homogeneous morphology compatible with that of mature trout lymphocytes, secreted supernatant 'factors', and were stained with rabbit antibodies to the ectodomain of recombinant trout T-cell receptor (TcR) beta-chain. Together with all the above mentioned properties, the presence of TcR sequences in the ADC cultures confirmed by the expression of alpha- and beta-chain TcR by nested PCR amplification and sequencing of the amplified bands, suggests that these ADC cultures contain trout T-like cells engaged in a VHSV response. These trout ADC cultures offer a first opportunity to further analyze fish anti-viral immunological responses.  相似文献   

12.
Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.  相似文献   

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Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.  相似文献   

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The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.  相似文献   

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The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.  相似文献   

16.
CD14, the leukocyte co-receptor for lipopolysaccharide (LPS), is important in the response of bovine polymorphonuclear neutrophil leukocytes (PMN) to Gram-negative bacteria. In other species, the expression of CD14 on the surface of PMN was shown to increase after exposure to inflammatory stimuli. These newly expressed molecules may originate from either an intracellular pool or through new gene expression. We sought to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and CD14's effect on secretion of the chemoattractants IL-8 and IL-1beta by PMN. Bovine PMN were incubated in RPMI for 20 h at 37 degrees C with LPS (1, 10, 100 microg/mL). IL-8 release increased with treatment of 1 microg/mL LPS, but decreased 41.5 and 95% at the 10 and 100 microg/mL concentrations of LPS, respectively. In contrast, shedding of CD14 from the surface of PMN only increased at the highest concentration of LPS (100 microg/mL). Secretion of IL-1beta was similar regardless of the LPS concentration used to stimulate PMN. The effect of PMN concentration (1 x 10(7), 2.5 x 10(7), 5 x 10(7), and 10 x 10(7)/mL) on CD14 cell surface expression and shedding of IL-8 and IL-1beta were also determined. Shedding of CD14 by PMN increased with increasing concentration of PMN after exposure to 0.1 and 10 microg/mL of LPS, while secretion of IL-8 decreased. IL-1beta increased at the highest concentration of PMN. The use of real time polymerase chain reaction showed that CD14 mRNA expression was not different between control and LPS-stimulated cells, indicating that the sCD14 came from either membrane bound CD14 or a preformed pool. Our results demonstrate that release of CD14 from PMN suppresses secretion of IL-8, and may be an important regulatory mechanism for controlling excessive migration of PMN into the bovine mammary gland.  相似文献   

17.
The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.  相似文献   

18.
A bioassay for bovine interleukin-1 (IL1) activity is described. The assay is based on the IL1-stimulated proliferation of a mouse T-lymphocyte cell line, D10(N4)M. Bovine mononuclear cells stimulated with lipopolysaccharide produce an interleukin-1-like activity which stimulated the growth of the D10(N4)M cell line in a dose-dependent manner. The stimulatory activity was neutralised by a combination of both anti-human IL1 alpha and anti-human IL1 beta sera. The quantity of IL1-like activity released from the mononuclear cells increased asymptotically with increasing lipopolysaccharide dose.  相似文献   

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为探讨鸡p15基因的生物学功能,试验构建了鸡p15基因的真核表达载体pcDNA3.1( )-p15,并转染到鸡MDV转化的淋巴细胞系MDCC-MSB1,应用G418筛选掉未转染的细胞,对存活细胞p15蛋白的表达、细胞增殖力、群体倍增时间、细胞周期和端粒酶的活性进行了检测。结果表明,与转染空质粒pcDNA3.1( )细胞相比,转染了p15基因的细胞稳定表达了p15蛋白;细胞的增殖受到了抑制,抑制率达45%~74%;群体倍增时间从27h延长至416h;流式细胞仪分析细胞周期发现,p15蛋白引起了细胞多停滞于G0/G1期,S和G2/M期细胞比例下降;端粒酶活性受到抑制。  相似文献   

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