Effects of Bacillus subtilis B10 spores on viability and biological functions of murine macrophages

This study was conducted to investigate the effects of Bacillus subtilis B10 spores on the viability and biological functions of murine macrophage. RAW 264.7 cells were stimulated both with and without B. subtilis B10 spores for 12 h. Then cell viability was determined to evaluate the cytotoxic effect of B. subtilis B10 spores to the cells, and the activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH), the production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and the secretion of inflammatory cytokines were measured to analyze the functions of macrophages. The results showed that B. subtilis B10 spores were not harmful to RAW 264.7 cells and they also strongly enhanced the activities of ACP and LDH (P < 0.01), remarkably increased NO and iNOS production (P < 0.01), and significantly stimulated the secretion of pro‐inflammatory cytokines, including tumor necrosis factor‐alpha (TNF‐α), interferon‐gamma (IFN‐γ), interleukin‐1 beta (IL‐1β), IL‐6, IL‐8 and IL‐12 (P < 0.01) while they reduced anti‐inflammatory cytokine IL‐10 (P < 0.01). The outcomes suggest that B. subtilis B10 spores are not only safe for murine macrophages, but also can activate these cells and enhance their immune function. The above findings suggest that B. subtilis B10 spores are potentially probiotic.     

Porcine interleukin 2: parameters of production and biochemical characterization

Interleukin 2 (IL2) or T cell growth factor (TCGF) has been characterized in a number of species but not in porcines. Porcine IL2 was detected in supernates (SN) of cultures of pig lymphocytes by: 1) the stimulation of the IL2-sensitive murine T cell line, CT6; 2) a costimulator assay involving porcine thymocytes; and 3) by the in vitro maintenance of antigen or mitogen-induced porcine lymphoblastoid cells. Porcine IL2 production by pig lymphocytes was induced by the mitogens Concanavalin A (Con A) Phytohemagglutiniin (PHA), and Pokeweed mitogen (PWM), but not by lipopolysaccharide (LPS). IL2 activity was demonstrated in the SN of mitogen-stimulated lymphocyte cultures as early as 24 hr after initiation of culture, reached peak levels at 48 hr, and decreased by 72 hr. Mitogens induced IL2 secretion by pig peripheral blood mononuclear cells, lymph node cells, and spleen cells, but not thymus cells. The cells responsible for IL2 production are presumptive T cells because: 1) they are nylon wool non-adherent; and 2) are non-surface-Ig bearing. In contrast, SN from cultures of surface Ig-positive cells had minimal IL2 activity. Porcine IL2 resembles rat and human IL2 in that it has an apparent molecular weight of approximately 15,000, and does not bind to DEAE-cellulose (DE-52) ion exchange columns equilibrated in 0.05 M sodium phosphate buffer (pH 7.6).     

Treatment of rats with heat killed cells (HKC) of Cryptococcus neoformans var. grubii induces cellular activation in spleen and lymphatic nodes

Our previous studies showed that the subcutaneous pretreatment of rats with heat killed cells of Cryptococcus neoformans (HKC) emulsified in complete Freund adjuvant (CFA) promotes protection against an intraperitoneal challenge with viable C. neoformans. In this model, an appropriate activation of adherent peritoneal cells after antigenic treatment is very important for the control of the infection. Here, we investigated the immune response developed in spleen and lymphatic nodes as a result of treatment with HKC-CFA, which might also contribute in the protective phenomenon of this treatment against cryptococcal infection. The results show that, compared with adjuvant alone, rats which received treatment with HKC-CFA presented a greater activation of adherent splenic cells, with up-regulation of major histocompatibility complex class II (MHC II) and CD86 expression and secretion of anticryptococcal metabolites. Furthermore, this treatment also induced an increase in the blastogenic response and the secretion of Th1 and Th2 cytokines by spleen cells in comparison with cells from CFA-phosphate-buffered saline (PBS) treated rats. On the other hand, lymph node cells from animals treated with HKC-CFA presented a rise in the expression of MHCII but not of CD86 with respect to control cells from rats treated with CFA-PBS. These cells also showed a high proliferative response and secretion of Th1-related cytokines, interleukin (IL)-12 and tumor necrosis factor (TNF). These results show that treatment of rats with HKC-CFA is able to induce an early immune response in secondary lymphoid organs, which may contribute to the protective effect induced by this treatment.     

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.     

Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) of Brucella abortus in cattle and mice

Brucella abortus vaccines composed of native cell envelopes or outer membrane proteins of smooth strain 2308 were compared with a vaccine (PG) composed of the insoluble residue of strain 2308 cell envelopes which had been extracted with hot sodium dodecyl sulfate. Vaccines were given by injection in an oil base adjuvant containing trehalose dimycolate and muramyl dipeptide or without adjuvant. Mice vaccinated with 30 micrograms native cell envelopes or PG and challenged 4 weeks later with virulent B. abortus strain 2308 displayed equivalent levels of protective immunity at 1 and 4 weeks post-infection. Heifers were vaccinated with 5 mg of antigens in adjuvant; PG was also administered without adjuvant. Humoral and cell mediated immune (CMI) responses were tested at monthly intervals. PG without adjuvant induced negligible immune responses. Native cell envelope antigens induced significantly higher titers of whole cell agglutinins over a 3-month period than did PG, although revaccination with PG in adjuvant enhanced the production of agglutinins and both vaccines induced antibodies to the O polysaccharide. Lymphocyte blastogenesis responses and delayed hypersensitivity reactions to porin and group 3 proteins were stimulated by both native and PG vaccines, and the magnitude of the responses did not differ significantly between the treatment groups. These vaccines were therefore comparable in their capacity to induce protective immunity in mice and CMI responses in cattle, whereas antibody responses induced by PG in cattle were generally lower. These findings provide a basis for evaluation of nonliving B. abortus vaccines in cattle.     

Negative regulation of interleukin 1β expression in response to DnaK from Pseudomonas aeruginosa via the PI3K/PDK1/FoxO1 pathways

Interleukin (IL)-1β is crucial for a wide range of inflammatory responses. Previously, we reported that IL-1β is produced in response to Pseudomonas aeruginosa-derived DnaK via NF-κB and JNK pathways; however, the signaling pathways that counter the process to maintain IL-1β homeostasis are unknown. Here, we show that DnaK-mediated expression of IL1β is increased markedly in macrophages upon blockade of PI3K/PDK1. This was verified by measuring released IL-1β protein. The negative effect of PI3K on IL-1β production was dependent on suppression of both NF-κB and JNK activation. Intriguingly, PDK1 (an underlying mediator of PI3K) acted as an upstream regulator for the activation of NF-κB, but downregulated JNK activation. Furthermore, production of IL-1β and activation of JNK were triggered by inhibition of phosphorylated FoxO1; phosphorylation of FoxO1 was controlled by PDK1 signaling in response to DnaK. Thus, IL-1β production is modulated by P. aeruginosa-derived DnaK via cross-talk between JNK and PI3K/PDK1/FoxO1 pathways.     

Antigen-nonspecific macrophage factors modulating the antibody response in vitro

Antibody response to an antigen involves the co-operation between three types of cells: macrophages, T cells and B cells. The cognate interactions between these cells play a fundamental role in the expression of a specific antibody response, but the last is modulated by antigen-nonspecific soluble factors produced either by macrophages or by T cells. Macrophages elaborate a spectrum of molecules modulating the function of lymphoid cells; among them are IL1 and prostaglandins of the E series, which are respectively enhancer and inhibitor of the antibody response in vitro. These molecules alter T cell and B cell activities through different mechanisms involving activation or inhibition of IL2 production, or alteration of cells surface antigens. However, the cellular events following the fixation of soluble factor on its receptors are not known.     

MicroRNA‐145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus‐induced mastitis in dairy cows

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus‐induced mastitis. In the present study, we overexpressed and suppressed miR‐145 to investigate the function of miR‐145 in Mac‐T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac‐T cytokines and in cell proliferation. We found that overexpression of miR‐145 in Mac‐T cells significantly reduced the secretion of IL‐12 and TNF‐α, but increased the secretion of IFN‐γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real‐time PCR (qRT‐PCR), Western blotting and luciferase multiplex verification techniques, we found that miR‐145 targeted and regulated FSCN1. Knock‐down of FSCN1 significantly increased the secretion of IL‐12, while the secretion of TNF‐α was significantly downregulated in Mac‐T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta‐miR‐145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.     

Recombinant bovine S100A8 and A9 enhance IL-1β secretion of interferon-gamma primed monocytes

Calgranulin A (S100A8) and B (S100A9) are found at high levels in inflamed tissue and have been associated with acute and chronic inflammatory disorders. Calgranulins are discussed as damage-associated molecular patterns (DAMPs). To analyze the role of calgranulins for inflammatory responses, bovine S100A8 and S100A9 were cloned, successfully expressed and FPLC-purified. Both molecules did not induce NF-κB activation in boTLR4-transfected HEK293 cells and stimulation of bovine monocytes with both proteins did not result in interleukin 1β (IL-1β) secretion or an upregulated mRNA expression of selected genes (IL1B, TNF, CXCL8, IL10, IL12). However, Interferon γ (IFN-γ) primed bovine monocytes released significantly higher amounts of IL-1β after stimulation with S100A8, S100A9, and co-stimulation with adenosine triphosphate (ATP). In IL-4/IL-13-primed monocytes, the IL-1β release was completely abrogated. The results imply that TLR4/MyD88/NF-κB-independent S100A8/A9-mediated activation of the inflammasome in cattle is favored in a Th1 environment and that S100A8 and S100A9 act as a DAMP in cattle.     

Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy

Interleukin (IL)-2 can induce large numbers of lymphokine-activated killer cells in peripheral blood lymphocytes (PBL), but IL-2 alone cannot induce proliferation of a large number of canine (c) PBL. We used the solid phase anti-CD3 antibody and soluble recombinant (r) IL-2 in order to establish a large scale culture method for cPBL. The number of lymphocytes seeded (3 x 10 (7)) increased to 1 x 10(9) after incubation for 10 days. The phenotype of cultured cPBL cells (after 2 weeks) showed a CD4(+) or CD8(+) predominant cell population. The cultured cell solutions were administered with physiological saline intravenously to each dog. After transfusion of the cultured cells, the cPBL counts, especially the number of CD4(+), CD8(+) and CD4(-)CD8 (-)(DN) cells increased significantly in the recipient dogs. Natural killer (NK) cells, gammadeltaT cells and B cells were considered to be present in the DN cell population. The NK cells and gammadeltaT cells showed no adverse reaction to the transfusion of the activated cPBL. Therefore, it is necessary to recognize the B cells present in the DN cell population by detecting CD21(+) cells. In conclusion, the bulk culture system of cPBL with rIL-2 and solid phase anti-CD3 antibody may be useful for the development of novel immunotherapy in dogs.     

Formation of interleukin-2 and interferon alpha by mononuclear leukocytes of swine after in vitro stimulation with different virus preparations

A new model was established to investigate the release of cytokines by porcine peripheral mononuclear leukocytes (MNL). Thereafter, this model is suitable to test paramunity inducers (biological response modifiers) and might replace animal models. The paramunity inducers Pind AVI and Pind ORF as well as other preparations of pox- and paramyxoviruses were employed. Production of both Interleukin 2 (IL2) and Interferon alpha (IFN alpha) was evaluated. Virus containing fluids induced an increase in IL2 activity (CTLL-2 proliferation assay) and IFN alpha (MDBK-cell line, VSV-virus) was noted. The release of IFN alpha and IL2 is caused by viral components. Pind AVI and Pind ORF induce production of IFN alpha in porcine MNL to a similar amount as ND-virus does. All virus preparations caused an increase in IL2 activity in supernatants of resting as well as mitogen-activated MNL without prior sensitization of the cells. The highest IL2-activity was measured after co-stimulation of the MNL with Concanavalin A and ND-virus.     

Comparison of the peripheral blood leukocyte population between Japanese Black and Holstein calves

Japanese black (JB) calves have greater susceptibility to infectious diseases compared to Holstein (Hol) calves. In order to clarify the differences in cellular immune status between JB and Hol calves, the leukocyte population and lymphocyte proliferative ability were analyzed. In total 200 healthy calves, 1 day to 14 weeks of age, were examined: 105 JB and 95 Hol calves. Lower numbers in peripheral blood and percentage in peripheral blood mononuclear cells of CD3⁺TcR1‐N12⁺ T cells and major histocompatibility complex class‐II⁺CD14^‐ B cells were observed in the JB compared to the Hol. The percentage of TcR1‐N12⁺CD25⁺ T cell in the JB was significantly lower than that of the Hol at 4–6, and 8–10 weeks. Interleukin (IL)‐2 sensitivity in the JB was lower than that in the Hol, and significant differences were observed in age groups of 6–8 weeks and 10–14 weeks. These findings indicated that the lower numbers of γδ T cells and B cells in the JB compared to the Hol might be associated with the specificity of the immune systems in JB calves.     

Validation of the Postprandial Interleukin-1β Response in Horses Using Equine-Specific Antibodies

Interleukin (IL)-1β is a commonly studied proinflammatory cytokine, with relevance to arthritis, obesity, aging, and other inflammatory diseases of horses. Evaluating protein concentrations in plasma is a useful measurement for research in these areas of equine health. The objective of this research was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) for equine IL1β and to compare concentrations with those previously published using an ELISA that is no longer available. The ELISA was assessed for linear parallelism and recovery using plasma from four healthy Standardbred mares. The assay was found to have linear parallelism for samples diluted 1:2, when the detection antibody concentration was 3 μg/mL. The average recovery of spiked IL1β was 98.9%. To compare concentrations, plasma was collected from six geldings at −0.5, 1, 2, and 4 hours after consumption of a meal high in starch and sugar (1.2 g/kg bodyweight). Consumption of 1.2 g of starch and sugar per kg of BW increased plasma IL1β concentrations 1 hour after feeding (P = .053). In conclusion, the commercially available ELISA is validated, with modifications, for use in equine plasma, and it detects a rise in plasma IL1β concentrations at 1 hour after meal consumption, a finding that is similar to previously reported data.     

Expression of mRNAs for interleukin-4, interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle

There is increasing evidence that inflammatory cytokines regulate corpus luteum (CL) function in many species. The purpose of the present study was to determine whether interleukin (IL)-4 and IL-6 are expressed in the porcine CL, and whether these cytokines influence porcine luteal steroidogenesis. The gene expressions of IL-4, IL-6 and their specific receptors were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of these cytokines on progesterone (P(4)), estradiol-17beta (E(2)) and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. IL-4 and IL-6 mRNAs were detected in the CL at all luteal stages. Furthermore, mRNAs of the receptors for IL-4 and IL-6 were clearly expressed in the CL throughout the estrous cycle. Real-time PCR analysis revealed that IL-6 receptor (IL-6R) mRNA expression was higher in the regressed CL (days 19-21 after ovulation) than in the CL at other stages (P<0.01). Exposure of cultured luteal cells obtained from mid-stage CL (days 8-11) to IL-6 (1-100 ng/ml), it inhibited P(4) and E(2) secretion by the cells (P<0.05). Although IL-4 (1-100 ng/ml) did not significantly alter P(4) secretion, it inhibited E(2) secretion by the cells (P<0.05). Neither IL-4 nor IL-6 had any effect on PGF2alpha secretion by the cells. These results suggest that IL-4 and IL-6 are locally produced in the porcine CL, and that they inhibit steroid production from luteal cells via their specific receptors. Collectively, both IL-4 and IL-6 may play roles in regulating porcine CL function throughout the estrous cycle.     

Adipocytes suppress differentiation of muscle cells in a co‐culture system

The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co‐culture system. The fusion index of C2C12 myoblasts co‐cultured with 3T3‐L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin‐1 and MuRF‐1 was higher in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes than the control. These results suggest that 3T3‐L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3‐L1 adipocytes induced the expression and secretion of IL‐6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co‐cultured with 3T3‐L1 cells in medium containing IL‐6‐neutralizing antibody than the control. Taken together, there is a possibility that adipocyte‐induced IL‐6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.     

Effects of Salmonella enterica serovar Typhimurium, or serovar Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis on chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2

Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-alpha (TNFalpha). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1h, followed by a wash. Media containing gentamicin was added and collected at 6h post-treatment. Compared to CTL, 10(8) ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization (P<0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFalpha mRNA expression by IPEC-J2 cells treated apically with 10(8) ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6h (P<0.05). However, BL increased IL8 mRNA at 1.5h (P<0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST (P<0.05) and BL (1.5 and 3.0 h; P<0.05), but not LR or SC. Only ST increased TNFalpha mRNA relative to CTL (P<0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL (P<0.0001), reduced by LR (P<0.05), and LR+ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST (P<0.0001), but blunted basolaterally in BL+ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.     

CpG oligodeoxynucleotides stimulate immune cell proliferation but not specific antibody production in rainbow trout (Oncorhynchus mykiss)

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.     

紫锥菊多糖对LPS损伤后IEC-6细胞分泌IL-1ɑmRNA的影响简

为了研究紫锥菊多糖对LPS损伤后IEC一6细胞分泌IL-1αmRNA的影响，以体外培养大鼠小肠上皮细胞为模型，进行细胞分组及收集，并给予LPS刺激造成内毒素损伤，根据GenBank中发布的IL-1α基因序列设计引物，以基因组R-NA为模板进行RT-PCR扩增，发现紫锥菊多糖对LPS刺激小肠上皮细胞产生的IL-1αmRNA的表达减少。     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Effect of lipopolysaccharide infusion on gene expression of inflammatory cytokines in normal horses in vivo

Horses are exquisitely sensitive to bacterial endotoxin and endotoxaemia is common in colic cases. In this study, gene expression of inflammatory cytokines was characterised in the blood of healthy horses following i.v. administration of lipopolysaccharide (LPS). Six horses received an LPS infusion and 6 controls received an equivalent volume of saline. Gene expression of genes encoding interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐8, and tumour necrosis factor‐α (TNF‐α) was quantified by real‐time PCR. Gene expression of all inflammatory cytokines was upregulated following administration of LPS. Interleukin‐1α, IL‐1β, IL‐8 and TNF‐α gene expression peaked at 60 min, while IL‐6 expression peaked at 90 min post LPS infusion. Interleukin‐1β and IL‐6 messenger RNA expression levels were above the baseline values 3 h post LPS infusion, whereas IL‐1α, IL‐8 and TNF‐α expression levels returned to baseline values by 3 h after LPS infusion. It was concluded that LPS infusion upregulated gene expression of inflammatory cytokines in the blood of healthy horses.