Bovine Interleukin-2 Research Articles

Cytotoxic T-lymphocyte antigen 4 and programmed cell death ligand 1 blockade increases the effectiveness of interleukin-15 immunotherapy in a bovine leukemia model.

Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4- and PD-L1-blocking algorithm. The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells. Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production. The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections.

Detection of C-Reactive Protein Using a Flexible Biosensor with Improved Bending Life

It is well known that regular monitoring of C-reactive protein is beneficial for the self-management of chronic diseases. Herein, a novel tattoo biosensor for regular measurement of C-reactive protein is presented. An innovative approach is presented to boost the life cycle of flexible biosensors in bending tests. Carbon nanotubes were deposited on the biosensor’s surface using electrophoretic methods. To assess suspension stability, a zeta potential measurement was performed. Optimized parameters of the electrophoretic procedure were evaluated through scanning electron microscopy imaging. This study shows that carbon nanotube coating increases the biosensor life-cycle by ten times (from 10 to 160 cycles). Using atomic force microscopy, the immobilization of a recognition element (aptamer) on the biosensor’s surface was verified. Furthermore, the biosensor’s performance was characterized using two electrochemical methods (cyclic voltammetry and impedance spectroscopy). The biosensor achieved a detection range of 0.02 − 0.8 ng ml−1. The sensor selectivity was examined with a solution of bovine serum albumin, immunoglobin E, interleukin 6, and tumor necrosis factor α. Finally, we carried out a test with the biosensor on spiked human serum samples to confirm that our proposed flexible biosensor can successfully monitor C-reactive protein levels in a range of clinical samples.

Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P < 1 × 10−5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.

Development of Foot and Mouth Disease Vaccine Formulation in Egypt by Using Silver Nanoparticles as Immunogenic and Adjuvant Corresponding author: Name: Rania Dweek, Tel: 01009377501 E-mail : raniadweek@yahoo.com

ABSTRACTObjective: To evaluate the effect of silver nanoparticles (AgNPs) with FMD inactivated oil adjuvant vaccine on immunological response of cattle.Study Design: Randomized clinical trial. Procedures: Silver nanoparticles (AgNPs) were prepared then added to locally produced inactivated trivalent FMD vaccine used in Egypt. Twenty cattle were classified into 4 groups (5 in each group).The first group was vaccinated by FMD trivalent inactivated oil adjuvant vaccine plus AgNPs (3ml subcutaneously), the second group was vaccinated by FMD trivalent inactivated oil adjuvant vaccine (3ml subcutaneously). The third group was vaccinated by FMD trivalent inactivated vaccine plus AgNPs as adjuvant (2ml subcutaneous). The fourth group was injected by AgNPs only (1 ml subcutaneous). Sera samples were collected from all animals groups for measuring humeral immunity by using serum neutralization test (SNT) and cell mediated immunity by using bovine Interleukin 6 (IL-6) at first ,second, third and fourth week for (IL-6 and SNT) and at 2 months and 4 months for SNT.Results: The size of AgNPs by TEM (Transmission Electron Microscope) is ranged from 5.8 to 28.51 nm. Antibody titer by SNT revealed that first group was the highest group reach to protective value at 2 weeks (1.538 log10) and reach to peak at 2 months (2.77 log10). Bovine IL-6 was revealed that animals vaccinated by AgNPs appear more quickly than that vaccinated by FMD inactivated oil adjuvant vaccine. Conclusion: The beneficial effect of AgNPs to potentiate both cellular and humeral immunity against FMD.

Characterization of bovine interleukin-2 stably expressed in HEK-293 cells

Interleukin 2 (IL-2) is a pleotropic cytokine and well-known as a T cell growth factor in immunology. It is now known to exert both immunostimulatory and immunosuppressive effects, optimizing immunological microenvironments for effector and regulatory T cell responses. The immunomodulatory role of IL-2 is critical for deciding whether or not T cell responses against specific antigens result in protection. We have established a mammalian cell line (HEK-293) stably expressing bovine IL-2 (boIL-2) (designated as HEK-293/boIL-2), using the piggyBac transposon system. The concentration of recombinant bovine IL-2 (rboIL-2) in the culture supernatant of HEK-293/boIL-2 reached 100 ng/ml on day 7 and showed similar proliferative activity to recombinant human IL-2 (rhuIL-2) for bovine peripheral mononuclear blood cells. Although rhuIL-2 has been often used to activate bovine T cells, our results indicate that characteristics of the T cell activation through rboIL-2 and huIL-2 appear slightly but significantly different. Interestingly, the rboIL-2/anti-boIL-2 monoclonal antibody (C5) (rboIL-2/C5) complex strongly induced proliferation of bovine NKp46+cells, natural killer (NK) cells, in vitro. This indicates that the rboIL-2/C5 complex could function as an IL-2 agonist specifically to increase the NK cell population, which in turn could enhance the activity of NK cells leading to protective immunity.

Protease inhibitors elicit anti-inflammatory effects in CF mice with Pseudomonas aeruginosa acute lung infection.

Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa-secreted proteases, in addition to host proteases, degrade lung tissue and interfere with immune processes. In this study, we aimed at evaluating the possible anti-inflammatory effects of protease inhibitors Marimastat and Ilomastat in the treatment of P. aeruginosa infection. Lung infection with the P. aeruginosa PAO1 strain was established in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knock-out C57BL/6 mice expressing a luciferase gene under control of bovine interleukin (IL)-8 promoter. After intratracheal instillation with 150µM Marimastat and Ilomastat, lung inflammation was monitored by in-vivo bioluminescence imaging and bacterial load in the lungs was assessed. In vitro, the effects of protease inhibitors on PAO1 growth and viability were evaluated. Acute lung infection was established in both wild-type and CFTR knock-out mice. After 24h, the infection induced IL-8-dependent bioluminescence emission, indicating lung inflammation. In infected mice with ongoing inflammation, intratracheal treatment with 150µM Marimastat and Ilomastat reduced the bioluminescence signal in comparison to untreated, infected animals. Bacterial load in the lungs was not affected by the treatment, and in vitro the same dose of Marimastat and Ilomastat did not affect PAO1 growth and viability, confirming that these molecules have no additional anti-bacterial activity. Our results show that inhibition of protease activity elicits anti-inflammatory effects in cystic fibrosis (CF) mice with acute P. aeruginosa lung infection. Thus, Marimastat and Ilomastat represent candidate molecules for the treatment of CF patients, encouraging further studies on protease inhibitors and their application in inflammatory diseases.

Development of a flow cytometry assay for bovine interleukin-2 and its preliminary application in bovine tuberculosis detection

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), poses a risk of infection for livestock, humans, and wildlife. An interferon (IFN)-γ release assay has been used with tuberculin skin tests to detect bTB; however, infected animals may still be missed. Previous studies have suggested that bovine interleukin-2 (BoIL-2) may act as a potential biological marker for the diagnosis of bovine infectious diseases. However, a detailed evaluation of IL-2 as a diagnostic target for bTB is lacking. Therefore, we established hybridoma cell lines that produced monoclonal antibodies (mAbs) recognizing the native BoIL-2 and developed a flow cytometry assay, based on the BoIL-2 mAbs, for detecting M. bovis-specific IL-2. Subsequently, the method was utilized for a preliminary investigation of bTB in cattle; significantly (P < 0.0001) more CD4+IL-2+ T cells were detected in infected cattle than in healthy animals when a specific mycobacterial antigen CFP-10–ESAT-6 fusion protein was used. Moreover, our method demonstrated high coincidence rates with the BOVIGAM® test and an IFN-γ flow cytometry assay for the diagnosis of bTB. These findings show that the present method may be useful for detecting bTB.

Expression of bovine interleukin 15 in Pichia pastoris and study on its biological activity: a T-cell activator

Interleukin 15 (IL-15) plays crucial role as stimulatory cytokine in generation and proliferation of CD8+ T memory cells. The present study was initiated to analyze the role of yeast-expressed bovine IL-15 in inducing the CD8+ T memory cells. The bIL-15 was cloned in pPICZαA expression vector. The expressed bovine IL-15 was purified by anion exchange chromatography and further characterized using SDS-PAGE and Western blotting. Biological activity of the purified protein was evaluated in vitro through induction of Bcl2 in IL-15 stimulated PBMCs was measured using qPCR and the phosphorylation of STAT3 was confirmed by immuno-staining of HEK273 cells. From the recent research studies, it was evident that the fatty acid oxidation catalyzed by Carnitine Palmitoyl Transferase 1a (Cpt1a) is a critical step during the conversion of effector CD8+ T cells to memory CD8+ T cells which is induced by IL-15. There were no research studies revealed the role of IL-15 on activating memory CD8+ T cells by influencing the Cpt1a in Bovines was documented; yet and in the present study, we evaluated that bovine IL-15 could induce the expression of Cpt1a in IL-15 treated bovine PBMCs and helps in memory cell induction.

In vivo relevance of polymorphic Interleukin 8 promoter haplotype for the systemic immune response to LPS in Holstein-Friesian calves

Interleukin 8, also known as CXC chemokine ligand 8 (CXCL8), is a critical chemokine in the recruitment of leukocytes to sites of infection and is a potent mediator of inflammation. We previously discovered 29 polymorphic sites in the promoter region of the bovine Interleukin 8 gene, which segregate into two distinct haplotypes, denoted IL8-h1 and IL8-h2. Population genetic analysis of these two haplotypes showed significant inter-breed differences in haplotype frequency, which is suggestive of selection acting at this locus. Furthermore functional characterisation identified that IL8-h2 was more active in mammary epithelial cells stimulated with the bacterial endotoxin, LPS. However, the in vivo relevance of these functional differences in the IL8 gene has not been ascertained. Therefore, in the current study, we tested the hypothesis that IL8 haplotype would result in variation in the systemic immune response to LPS challenge in Holstein-Friesian (HF) calves. A Taqman assay was designed to genotype both Jersey and HF calves, from which 20 healthy HF calves (representing IL8-h1 and IL8-h2) from the same farm were subjected to LPS stimulation via jugular venepuncture (100ng/kg). Systemic immune profiling was subsequently performed up to 216h post-challenge. Haematological analysis showed perturbations in leukocyte populations of cells but only the lymphocyte response was significantly different between IL8-h1 and IL8-h2. IL8 expression levels were significantly different between haplogroups, at both the gene expression and protein levels (P<0.05). Circulating neutrophils were subsequently purified from each haplogroup to measure potential haplotype specific effects on neutrophil migration and bacterial killing but no significant differences were detected, which is likely due to the low circulating levels of IL8. We conclude that IL8 haplotype significantly affects IL8 expression profile in response to bacterial endotoxin in vivo, and the significant increase in IL8 in IL8-h2 calves supports our earlier findings in vitro. Genetic variation at the IL8 locus therefore explains a proportion of the inter-breed and inter-individual variation in immunity between neonatal calves which is likely to influence their resistance to infection.

Expression of biologically active bovine interleukin 7 and evaluating the activity in vitro.

Aim:Interleukin 7 (IL-7) is a ϒc family cytokine involved in the homeostatic proliferation and maintenance of immune cells. In the present study, we report the expression of bovine IL-7 (bIL-7) in Escherichia coli and evaluated for its biological activity.Materials and Methods:The sequence coding for bIL-7 (mature protein) was amplified from primary bovine kidney cell culture and cloned into pET28-a vector and expressed in E. coli (BL 21 DE3). The expressed protein was purified by nickel-nitrilotriacetatechromatography, and the reactivity of the protein was confirmed by western blotting using monoclonal antibodies raised against human IL-7. The biological activity of expressed bIL-7 was evaluated by analyzing its effect on the expression of anuclear factor for activated T-cells c1 (NFATc1), B-cell lymphoma 2 (Bcl2), suppressor of cytokine signaling 3 (SOCS3) molecules in bovine peripheral blood mononuclear cells (PBMCs) by quantitative polymerase chain reaction. Ability of the expressed protein was also analyzed by its effect on phosphorylating signal transducer and activator 3 (STAT3) molecule by immunostaining in human embryonic kidney cells 293 (HEK293) cells.Results:The bIL-7 was able to induce the expression of Bcl2 and NFATc1expression in bovine PBMCs by 7 and 5-folds, respectively, whereas a 2-fold decrease was observed in the case of SOCS3 expression. Immunostaining studies in HEK293 cells using antihuman phospho-STAT3 showed activation and nuclear translocation of STAT3 molecule on bIL-7 treatment.Conclusion:bIL-7 gene was successfully amplified, cloned, and expressed in a prokaryotic expression system. The biological activity study showed that the E. coli expressed bIL-7 protein is biologically active. Considering the role of IL-7 in T-cell homeostasis and memory cell generation, this molecule can be used for enhancing the vaccine response and that has to be proved by further experiments.

Expression of bovine interleukin 15 and evaluation of its biological activity in vitro.

Background/Aim:Recent studies have shown that interleukin-15 (IL-15)is a critical factor for the development and proliferation of CD8+ memory T cells. The aim of present study is to study the role bovine IL-15 (bIL-15)in activation pathway of bovine CD8+ T cells if any, which will be useful in designing the adjuvant to increase the duration of immunity of the vaccine preparations.Materials and Methods:Coding region of bIL-15 (489) was amplified from cDNA of lipopolysaccharide-induced bovine peripheral blood mononuclear cells (PBMCs) using gene specific primers and cloned into pcDNA3.1+. Mature length of bIL-15 was amplified using gene specific primers and cloned into pET32a for expression studies. Expressed fusion protein was purified using Ni-Nitrilotriacetic acid agarose affinity chromatography and analyzed by SDS-Polyacryamide gel electrophoresis (PAGE) and western blotting. Biological activity of purified protein was analyzed by quantitative Polymerase Chain Reaction (qPCR) for an increase in levels of Bcl2, STAT3 and STAT5a using cDNA synthesized from RNA of PBMCs induced with different concentrations of purified bIL-15. Role of IL-15 in inducing memory CD8+ T cells was analyzed by qPCR for increase in the level of Carnitine Palmitoyl Transferase 1a (CPT1a) using cDNA synthesized from RNA of PBMCs induced with different concentrations of purified bIL-15.Results:Bovine IL-15 was amplified and analyzed by agarose gel electrophoresis, which showed a specific product of ~490bp, mature sequence was amplified using full-length as a template to get a product of ~350bp. The protein was expressed, purified and analyzed by SDS-PAGE and Western blotting, which showed a specific product of 32kDa. Biological activity of purified bIL-15 fusion protein showed an increase in levels of Bcl2, STAT3 and STAT5a with 5 fold, 9 fold, and 10 fold increases as analyzed by qPCR, respectively. Role of IL-15 in inducing memory T cells showed an increase in expression level of CPT1a at 2.5 fold increase as compared to control cells.Conclusion:Bovine IL-15 has been successfully cloned and expressed in our work, and the biological activity shows that the purified fusion protein is biologically active. As there is an increase in levels of CPT1a an enzyme critical for survival of memory T cells, IL-15 can be used for increase in the memory response, which can be used as an adjuvant with viral vaccines for increasing the immunity.

Polyfunctional CD4 T cells in the response to bovine tuberculosis. (VET2P.1034)

Abstract Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) and play relevant roles in several chronic infections, including human TB and HIV. However, the assessment of this response in bovine infections was not feasible due to the lack of monoclonal antibodies that recognize bovine IL-2. Recently, an antibody specific for bovine IL-2 enabled the evaluation of polyfunctional T cells in cattle. The objective of the present study was to access antigen-specific polyfunctional ex vivo responses after aerosol Mycobacterium bovis infection of cattle. Peripheral blood mononuclear cells (PBMCs) were collected from infected cattle and stimulated with rESAT-6:CFP-10 (E:C), media or pokeweed (PWM) for 16 hours. After stimulation, the expression of CD4, CD45RO, CCR7, IL-2, IFN-γ, TNF-α and cell viability were evaluated. The ex vivo response to E:C consisted of 63% effectors (CD4+ CD45RO- CCR7-), 32% effector memory (CD4+ CD45RO+ CCR7-), and 9% central memory (CD4+ CD45RO+ CCR7+) phenotypes. In regard to the cytokine profile, 70% of cells producing cytokines expressed both IFN-γ and TNF-α, 31% expressed all three cytokines, 6% expressed both TNF-α and IL-2, and 3% expressed IL-2 and IFN-γ. Cells producing only IFN-γ, IL-2, or TNF-α represented, 5%, 8% and 1%, respectively. These findings demonstrate that the polyfunctional response consists mainly of effector cells co-producing IFN-γ and TNF-α.

Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis

We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.

In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter–luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter–luciferase construct is transiently and robustly activated 3–5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter–luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions.

Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor 488 SAA as well as of other well established CD36 ligands was increased 5-10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10-50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60-75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36(-/-) rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-kappaB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-kappaB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.

Construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18

Selection of potent cytokine adjuvants is important for the development of Staphylococcus aureus DNA vaccines. Several potential cytokines have been proven to induce enhanced immune responses in animal models and clinical tests. There is still no reported use of IL18 as an adjuvant to design DNA vaccines against S. aureus. In this study, we cloned the main fibronectin binding protein gene (a fragment from clumping factor A, ClfA 221–550) of S. aureus and bovine interleukin 18 (bIL18). Then recombinant plasmids were constructed based on the eukaryotic expression vector pVAX1 with or without bIL18. Indirect immunofluorescence assays in transfected HeLa cells indicated that the recombinant DNAs (rDNAs) could be expressed correctly and had antigenicity. BALB/c mice were used as experimental models to examine the immunogenicity of rDNAs in vivo. The ClfA 221–550 rDNA provoked antibody production. The bIL18 rDNA induced production of the Th1 type cytokines IL2 and IFNγ, and ClfA 221–550 and bIL18 synergistically stimulated T-lymphocyte proliferation. The data demonstrated that bIL18 is a potent adjuvant that could be used to enhance cellular immunity.

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins

The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein.

Interleukin-1β infusion in bovine mammary glands prior to challenge with Streptococcus uberis reduces bacterial growth but causes sterile mastitis

Dairy cows are especially vulnerable to intramammary infection by the bacterial pathogen Streptococcus uberis in the dry period. Use of immunotherapeutic agents at drying off could increase cellular defences in the gland and prevent establishment of new S. uberis infections. This study investigated the potential of infusing recombinant bovine interleukin-1 beta (rbIL-1beta) in the mammary glands as a prophylactic agent against subsequent intramammary challenge with S. uberis in the early dry period. Immediately after the last milking at commencement of the dry period, one cow from each of 10 monozygous twinsets was infused with 10 microg of rbIL-1beta in two quarters and the other twin was infused with the carrier agent, sterile phosphate buffered saline. Twenty-four hours later, the quarters were infused with 10(3) colony-forming units (CFU) of S. uberis. Bacteriology, somatic cell count (SCC), concentrations of specific cytokines and antibody responses were monitored in mammary gland secretions and sera for the next 21 days. Infusion of rbIL-1beta into mammary glands at commencement of the dry period was associated with less new S. uberis intramammary infections, as determined by the number of quarters with bacterial growth. However, high SCC in quarters following infusion of rbIL-1beta masked the full beneficial effect of this procedure.

Heparan Sulfate Regulates Self-renewal and Pluripotency of Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.