Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

Influence of persistent canine distemper virus infection on expression of RECK, matrix-metalloproteinases and their inhibitors in a canine macrophage/monocytic tumour cell line (DH82)

A morbillivirus infection of tumour cells is known to exert oncolytic activity, but the mechanism of this inhibitory action has not been well defined. Matrix metalloproteinases (MMPs) are important enzymes degrading the extracellular matrix and are often upregulated in malignant neoplasms. Recent studies have demonstrated that RECK may potently suppress MMP-2 and -9 activity, thus inhibiting angiogenesis and metastasis. In this study, real time quantitative polymerase chain reaction (RT-qPCR) was used to determine the effect of persistent infection with canine distemper virus (CDV) infection on the expression of MMPs and their inhibitors (TIMPS) in a canine macrophage/monocytic tumour cell line (DH82). The activity of proMMP-2 and proMMP-9 was also verified zymographically. Following CDV infection, MMP-2, TIMP-1 and TIMP-2 were down-regulated, while RECK was upregulated. These findings suggest that CDV infection restores RECK expression in tumour cells and may interfere with the intracellular processing of MMPs and TIMPs, thus possibly influencing tumour cell behaviour beneficially for the host. However, this needs to be verified in in vivo studies.     

地锦草总黄酮对小鼠免疫功能及细胞因子mRNA表达影响

探讨地锦草总黄酮对小鼠免疫功能及细胞因子IL-2、IL-12、IFN-γ、TNF-αmRNA表达影响。将小鼠随机分成4组：地锦草总黄酮高剂量组（H组）、中剂量组（M组）、低剂量组（L组）和纯水对照组（C组）,连续灌胃9 d后,检测其单核巨噬细胞吞噬功能、2%绵羊红细胞诱导的小鼠迟发型变态反应、肝脏指数和脾脏指数,并用逆转录酶-聚合酶链式反应的方法检测脾脏IL-2、IL-12、IFN-γ和TNF-αmRNA表达水平。结果显示,地锦草总黄酮3个剂量组脾脏指数、廓清指数（K）、吞噬指数（α）、24 h足跖增厚值均显著高于C组;M组和H组肝指数显著高于C组（P〈0.01）;3个剂量组均能提高IL-2、IL-12、IFN-γ和TNF-αmRNA的表达水平,与C组相比,H组最显著。试验结果表明,地锦草总黄酮能有效提高机体的免疫功能及IL-2、IL-12、IFN-γ和TNF-αmRNA表达。     

紫花地丁总黄酮体外抗炎活性研究

试验研究了紫花地丁总黄酮（TFV）对脂多糖（LPS）诱导的小鼠RAW264.7巨噬细胞活力、细胞中炎症介质含量以及相关基因表达的影响,以期探讨其体外抗炎活性的作用。试验采用MTT法筛选出TFV对小鼠RAW264.7巨噬细胞活力具有促进作用的最佳添加浓度;用酶联免疫吸附法（ELISA）检测了TFV对LPS诱导的小鼠RAW264.7巨噬细胞释放到细胞培养液中NO、肿瘤坏死因子α（TNF-α）、白介素1β（IL-1β）、白介素6（IL-6）含量的影响;运用实时荧光定量PCR法检测了TFV对LPS诱导的炎性小鼠RAW264.7巨噬细胞TNF-α、诱导型一氧化氮合酶（iNOS）和环氧合酶2（COX-2）相对表达水平的影响;研究并分析了TFV的体外抗炎活性。试验结果表明,TFV在5~50 μg/mL浓度范围内能提高小鼠RAW264.7巨噬细胞的活力（P<0.05）;与LPS模型组比较,TFV能显著降低LPS诱导的小鼠RAW264.7巨噬细胞产生NO、TNF-α、IL-6、IL-1β的含量,并能显著降低LPS诱导的小鼠RAW264.7巨噬细胞内TNF-α、COX-2等炎症因子的mRNA表达量（P<0.05）。综上,TFV能显著下调LPS诱导的小鼠RAW264.7巨噬细胞IL-1β、IL-6、TNF-α等细胞因子的释放量和下调TNF-α、COX-2 mRNA的表达量,说明抑制促炎性细胞因子基因的表达可能是实现其抗炎作用的原因之一。     

Evaluation of biomarkers for osteoarthritis caused by fragmented medial coronoid process in dogs

The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.     

Kinetics of IL-6 and TNF-α changes in a canine model of sepsis induced by endotoxin

Sepsis is a major cause of death in veterinary medicine, although a better prognosis can result from an early diagnosis. To speed the diagnosis, the biomarkers TNF-α and IL-6 can provide valuable information regarding systemic inflammatory response. The purpose of this study was to investigate the changes in cytokine levels in an experimental model of sepsis using ELISA and real-time PCR. Ten adult Beagles were studied; seven received an IV bolus of high dose lipopolysaccharide solution (1mg/kg) to induce sepsis. The remaining three beagles were the control group. Blood samples were collected before and 1, 3, 6, 12, 24 and 48 h after administering LPS. Serum IL-6 level peaked at 3h (1.89 ± 0.10 ng/ml) and serum TNF-α peaked at 1h (1.11 ± 0.01 ng/ml). The expression of IL-6 mRNA in peripheral blood mononuclear cells (PBMC) increased 62-fold compared to the control group at 1h; TNF-α mRNA increased by 4.5-fold at 1h. The expressions of IL-6 and TNF-α mRNA in PBMCs changed more rapidly than serum IL-6 and TNF-α concentrations. In addition, TNF-α mRNA levels in PBMCs remained elevated longer than serum TNF-α. Our study establishes the basis for future work aimed at a better understanding of the systemic inflammatory response to infection and sepsis in canine patients.     

β-羟丁酸抑制脂多糖诱导奶牛中性粒细胞核因子-κB信号通路的激活

高酮血症造成奶牛中性粒细胞先天免疫机能受到抑制,本研究探讨 β-羟丁酸(BHBA)是否抑制脂多糖(LPS)诱导的奶牛中性粒细胞核因子-κB(NF?κB)信号通路的激活.分离健康奶牛中性粒细胞,采用LPS(100 ng/mL)和不同浓度(0.5、1.0、2.0和4.0 mmol/L)BHBA作用于中性粒细胞,收集细胞,应...     

Activity of matrix metalloproteinase-2 (MMP-2) in canine oronasal tumors

Activity of matrix metalloprotease-2 (MMP-2) and the expression of its related molecules were examined in spontaneous canine oronasal tumors. Tissue samples from melanoma and squamous cell carcinoma possessed higher MMP-2 activity, as shown in gelatin zymography, in comparison with acanthomatous epulis and nasal adenocarcinoma. Regional lymph node invasion and distant metastases were more frequently observed in the MMP-2 positive cases. There were no significant differences by RT-PCR examination in the expression of the genes encoding MMP-2, MT1-MMP and TIMP-2 among the tumor histological types. However, the MMP-2/TIMP-2 ratio showed a significantly higher level of the genes in the malignant oral melanoma and squamous cell carcinoma. The MMP-2/TIMP-2 ratio was also positively correlated with MMP-2 activity in gelatin zymography. These results indicate that the MMP-2/TIMP-2 ratio may be of value in evaluating the prognosis in canine oronasal cavity tumors.     

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

ABSTRACT: Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs.     

Matrix Metalloproteinase (MMP)-2 and MMP-9 Activity in the Canine Uterus Before and During Placentation

The aim of the present study was to demonstrate the presence and localization of MMP-2 and -9 by means of RT-PCR and immunohistochemistry (IHC) within the canine uterus from the pre-implantation stage until mid-gestation and to determine MMP-2 and -9 activities by means of zymography. For this purpose, samples of the uterus and salpinx from bitches were obtained after ovariohysterectomy. Pre-implantation stages (5-12 days after mating, n = 11) were determined by verifying embryos after flushing the uterus. Further groups were determined as implantation (15-19 days after mating, n = 9), post-implantation (20-30 days after mating, n = 9) and placental stages (30-45 days after mating, n = 3). A non-pregnant group (17-30 days after mating, n = 4) served as control. MMP-2 and -9 positive cells were detected in all specimens from pregnant and nonpregnant bitches, however, with different distributions. MMP-2 was present in endothelium and smooth muscles of blood vessels and the myometrium of pregnant and nonpregnant bitches, additionally in the surface epithelium of the oviduct. The latter also stained positive for MMP-9. During placentation, MMP-2 was detected mainly in fetal blood vessels and trophoblastic cells. Higher MMP-2 activity was observed in the endometrium and myometrium of all pregnant groups compared with the nonpregnant group (p < 0.05). The pregnant groups did not differ significantly from each other (p > 0.05). MMP-9 was present in blood vessels, smooth muscle cells and epithelia, such as maternal surface epithelial cells, uterine crypts and glands. During placentation, the deep uterine glands and the epithelium of the glandular chambers were immunoreactive to MMP-9. Highest MMP-9 activities were reached in the endometrium of the pre-implantation group (23.2% of total MMP-9) and placental parts (33.3%).     

孕酮对LPS诱导小鼠腹腔巨噬细胞表达TNF-α、IL-1β、TLR4、CD14和MD2的影响

先分离培养小鼠腹腔巨噬细胞,经差速贴壁法纯化后,随机分为6组：空白对照组、0.5mg/L脂多糖（LPS）组、10-6 mol/L孕酮（P4）组、LPS＋10-5 mol/L P4组、LPS＋10-6 mol/L P4组、LPS＋10-7 mol/L P4组。各组在处理12、24h分别提取上清液,ELISA法测TNF-α和IL-1β的含量;各组在处理24h分别提取细胞总RNA,用RT-PCR法测TLR4、CD14、MD2mRNA的表达。结果显示,处理12、24h,0.5mg/L LPS组TNF-α和IL-1β的含量均极显著高于对照组（P〈0.01）;10-6 mol/L P4组与对照组差异不显著（P〉0.05）;LPS＋10-5 mol/L P4组极显著低于对照组（P〈0.01）;LPS＋10-6 mol/L P4组显著低于对照组（P〈0.05）;而LPS＋10-7 mol/L P4组TNF-α的表达差异不显著（P〉0.05）,IL-1β的表达差异显著（P〈0.05）。说明P4可降低LPS刺激小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,且呈剂量依赖关系。LPS单独处理,TLR4和CD14mRNA的表达极显著高于对照组（P〈0.01）;10-6 mol/L P4单独处理与对照组无显著差异（P〉0.05）;分别添加1-5、10-6、10-7 mol/L P4组均极显著降低LPS诱导TLR4和CD14mRNA的表达（P〈0.01）,而MD2mRNA的表达差异不显著（P〉0.05）。说明P4可极显著降低LPS刺激小鼠腹腔巨噬细胞TLR4和CD14mRNA表达,但对MD2mRNA表达影响不显著。结果显示,P4能抑制LPS刺激的小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,此过程与细胞TLR4和CD14表达下降相关,而与MD2的表达无关。     

An immunohistochemical study of the role of matrix metalloproteinases and their tissue inhibitors in chronic mitral valvular disease (valvular endocardiosis) in dogs

While the pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, alterations in the activity of specific metalloproteinase enzymes and their inhibitors within the valve stroma are suspected of having a role. This study describes the immunohistochemical distribution pattern of matrix metalloproteinase (MMP) types 2, 9 and 14 and their tissue inhibitors, termed tissue inhibitors of metalloproteinase (TIMP), types 2 and 3, in normal canine mitral valves (MVs) (n=10) and in dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal MVs, MMP-2 and -14, and TIMP-2 were expressed in isolated stromal cells. Tissue inhibitor of metalloproteinase-3 exhibited moderate intracellular and mild extracellular expression. With increasing severity of CVD, the expression of MMP-2 decreased. The number of stromal cells expressing MMP-14 increased, predominantly in the margins of the nodular lesions. Tissue inhibitor of metalloproteinase-2 and -3 expression increased both intra- and extracellularly. Matrix metalloproteinase-9 was not detected in normal or diseased valves. In conclusion, CVD was characterised by alterations in the distribution and intensity of valvular MMP and TIMP expression, suggesting that depressed catabolism and the accumulation of extracellular matrix components within affected valves contributes to their structural alteration and consequent loss of function.     

家蚕蛹虫草菌丝体水提物对CCl4诱导小鼠肝脏和免疫功能损伤的保护作用

为发掘家蚕蛹虫草的药用价值,研究家蚕蛹虫草菌丝体水提物对CCl4诱导的小鼠肝脏及免疫功能损伤的保护作用。将ICR雄性小鼠随机分为正常对照组、模型组和家蚕蛹虫草菌丝体水提物低、高剂量组[0.2、0.4 g/(kg.d)],除正常对照组外各组均按剂量30 mg/kg腹腔注射CCl4造模。测定各组小鼠血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)活性和免疫因子白细胞介素2(IL-2)、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)的水平,结果表明家蚕蛹虫草菌丝体水提物可极显著提高模型小鼠血清中的IL-2、IFN-γ水平(P<0.01),并极显著降低AST、ALT活性及TNF-α含量(P<0.01)。研究结果显示家蚕蛹虫草菌丝体水提物对CCl4诱导的肝损伤具有明显的保护作用,并通过促进IL-2、IFN-γ的分泌,进而降低TNF-α的水平,调节CCl4模型小鼠的免疫功能,这也是其发挥抗肝脏纤维化作用的机制之一。     

The antagonist activity of lipid IVa on the stimulation by lipid A of TNF-alpha production from canine blood mononuclear cells

Lipid A, the active component of lipopolysaccharide (LPS), exists in the outer membrane of Gram-negative bacteria and binds to the Toll-like receptor 4 (TLR4) and MD-2 complex. On the other hand, the synthetic precursor of Escherichia coli lipid A, tetraacylated lipid IVa, is an agonist for TLR4 and MD-2 complex in murine, equine and feline cells but is an antagonist for lipid A in human cells. The aim of the study was to examine the function of canine Toll-like receptor 4 (TLR4) and MD-2 complex on canine blood mononuclear cells (BMC), by analyzing lipid A- or lipid IVa-induction of TNF-α production from these cells in order to understand canine innate immune system. After 5-h culture of canine BMC with lipid A (lipid A culture) or lipid IVa (lipid IVa culture), the TNF-α, as determined by ELISA, had increased in the supernatants of the lipid A cultures in a dose-dependent manner, whereas the TNF-α was undetectable in supernatant of lipid IVa-treated cultures. The TNF-α was statistically significantly different between the lipid A and lipid IVa cultures (100 and 1000 ng/ml). TNF-α production from canine BMC was inhibited, in a lipid IVa-dose-dependent manner, when the BMC were pre-cultured with lipid IVa for 60 min and then cultured with lipid A for 5h, while in control BMC cultures production if TNF-α was unchanged. These results indicate that the TNF-α production stimulated by lipid A was competed out by pre-exposing the BMC to lipid IVa. Thus, lipid A is an agonist for TNF-α production in canine BMC, whereas lipid IVa appears to be an antagonist against this lipid A stimulation of canine BMC.     

9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.     

Cytokine induction by immunostimulatory DNA in porcine PBMC is impaired by a hairpin forming sequence motif from the genome of Porcine Circovirus type 2 (PCV2)

Porcine Circovirus type 2 (PCV2) can cause postweaning multisystemic wasting syndrome (PMWS) in young pigs with severe immunosuppression as a major characteristic of the disease complex. Despite the dramatic involvement of the immune system, the interaction between PCV2 and the host is until date not well understood. The DNA genome of PCV2 contains sequences that in synthetic form (oligodeoxyribonucleotides; ODNs) can act immunomodulatory on porcine peripheral blood mononuclear cells (poPBMCs) in vitro. One such sequence (ODN PCV2/1) acts inhibitory on interferon (IFN)-α production induced by immunostimulatory DNA but not that induced by RNA, and the inhibitory activity is dependent on secondary structure formation. In the present study, the characteristic of ODN PCV2/1 was examined further by altering the nucleotide sequence to disrupt hairpin structure formation but still enable multimer structures through G-tetrads. This modification resulted in loss of IFN-α-inhibitory activity of the ODN and thus indicated the importance of hairpin structures. In addition, ODN PCV2/1 was compared to another inhibitory ODN (IRS 869) previously used in human and murine cells. In contrast to ODN PCV2/1, ODN IRS 869 did not inhibit IFN-α production induced by class A ODN 2216 but was a more efficient inhibitor of IFN-α production induced by plasmid DNA than ODN PCV2/1. In cultures induced by the RNA stimulator Poly I:C, however, a strong synergistic IFN-α stimulatory effect was seen in combination with ODN IRS 869. These results indicate that ODN PCV2/1 and ODN IRS 869 function through separate mechanisms to affect cytokine production by immune cells. The effect of ODN PCV2/1 was studied further by monitoring the expression of mRNA for IFN-α, IL-12p40, IL-10, IL-6, IFN-γ, IL-1β, TGF-β, and TNF-α in cultures of poPBMC stimulated with ODN 2216 or Poly I:C. Results from qPCR analyses showed that ODN PCV2/1 clearly inhibited the expression of IFN-α, IL-12p40, IL-10 and IL-6 when induced by ODN 2216, but did not seem to affect any of the cytokines examined when induced by Poly I:C. Initial studies using confocal microscopy and fluorochrome labelled ODNs indicate that ODN 2216 and ODN PCV2/1 co-localize in subpopulations of poPBMC.