Differentiation of porcine dendritic cells by granulocyte-macrophage colony-stimulating factor expressed in Pichia pastoris

Dendritic cells (DC) are potent inducers of acquired immunity due to their ability to present antigens in the context of a costimulatory environment and consequently serve an essential role in vaccine efficacy. Strategies to enhance their function, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 treatment to induce DC differentiation from peripheral blood monocytes, may therefore be useful as vaccine adjuvants. We now have evaluated the effect of recombinant GM-CSF on the differentiation of DC in swine. GM-CSF mRNA was readily detected in porcine splenocytes, with increased levels following treatment of the cells with ConA and LPS. Porcine GM-CSF was cloned and expressed in the methylotrophic yeast, Pichia pastoris, as a glycosylated protein that induced proliferation of porcine bone marrow cells. P. pastoris-derived GM-CSF induced expression of antigen presenting (MHC class II) and costimulatory (CD80-CD86) molecules and enhanced antigen presenting cell (APC) function consistent with the induction of functional DC. Thus, recombinant GM-CSF produced by P. pastoris may be a potent adjuvant for swine vaccines.     

粒细胞-巨噬细胞集落刺激因子基因佐剂对DNA疫苗的免疫调节作用

DNA疫苗具有制备简单,不需要蛋白质的合成、提取与纯化,免疫效果维持时间长,稳定性好,易保存等特点。但由于种与种之间免疫遗传差异,DNA疫苗往往对大动物特别是对哺乳动物的免疫效果不理想。细胞因子常作为基因佐剂用以增强DNA疫苗的免疫原性。粒细胞-巨噬细胞集落刺激因子(GM-CSF)是一个具有多项潜能的造血生长因子,在免疫反应中具有重要作用,编码GM-CSF的质粒能增强DNA疫苗的免疫效果。本文就GM-CSF的分子结构、对增强疫苗免疫原性、协同其他因子作用等方面作一综述。同时介绍了基因佐剂与目的基因表达质粒的构建方案。     

PGM-CSF对手工克隆猪生产效率的影响

为了优化猪的手工克隆技术方案,从而提高猪手工克隆效率,通过比较不同浓度(2和10 ng/m L)的猪粒细胞-巨噬细胞集落刺激因子(PGM-CSF)对孤雌胚胎和克隆胚胎的囊胚发育率及囊胚细胞数的影响,并将PGM-CSF处理过的手工克隆囊胚进行移植,比较对照组和试验组的妊娠率、产仔率及克隆整体效率。结果显示:用10 ng/m L PGM-CSF处理重构胚后,试验组胚胎细胞数统计略高于对照组((47.74±3.88)vs(43.69±3.72)),囊胚率高于对照组((39.10±0.01)%vs(36.27±0.02)%),但差异均不显著(P0.05);试验组10只受体中有5只怀孕且分娩,而对照组10只受体只有1只怀孕且分娩;试验组出生仔猪存活率略高于对照组(36.36%vs 30.00%),克隆总效率略高于对照组(0.37%vs 0.30%),但没有显著差异(P0.05)。综上所述,PGM-CSF有利于提高猪手工克隆胚胎整体发育能力,有提高猪手工克隆对受体的利用率的潜在可能性。     

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.     

Bovine recombinant granulocyte-macrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro

Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbGM-CSF)/ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxicity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbGM-CSF significantly increased (P less than 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbGM-CSF-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.     

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor on bovine peripheral blood neutrophil functions in vitro and in vivo

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) on bactericidal activity of bovine peripheral blood neutrophils in vitro and in vivo were studied. In in vitro experiment, bovine blood neutrophils were cultured for 9 hr in media containing 0.005, 0.05 or 0.5 microg/ml of rboGM-CSF. Neutrophils treated with rboGM-CSF showed significantly higher luminol-dependent chemiluminescence (LDCL) than control cells. In in vivo experiment, neutrophils isolated from cows injected 5.0 microg/kg of rboGM-CSF showed significantly higher Nitrobluetetrazolium (NBT) reduction value than that from control cows 24 hr post injection. Total leukocyte counts of cows injected rboGM-CSF sharply decreased 6 hr post injection and recovered to normal level 2 days post injection. Body temperature of these cows rose 6 hr post injection and back to normal level at 24 hr post injection. It was suggested that rboGM-CSF enhanced bactericidal activity of bovine neutrophils both in vitro and in vivo.     

Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and mammary gland neutrophil function in vitro.

Modulation of peripheral blood and mammary gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 microgram/mL rBoGM-CSF. Neutrophils treated with rBoGM-CSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections.     

鸡胚接种鸡白介素18重组质粒对IBDVDNA疫苗免疫增强作用的研究

用鸡白细胞介素18(ChIL-18)重组质粒(pCI-ChIL-18)接种18胚龄SPF鸡胚,利用适时荧光定量PCR方法对试验鸡不同时间点的动态变化进行检测.检测结果表明,胚胎接种pCI-ChIL-18使ChIL-18在鸡血液中早期持续高浓度存在,能促进鸡脾脏和法氏囊中ChIL-18基因的大量表达.用pCI-ChIL-18与传染性法氏囊病病毒(IBDV)多聚蛋白DNA疫苗分别免疫18胚龄SPF鸡胚,设1日龄雏鸡肌肉免疫对照组和空白对照组,2周龄时二免,6周龄时用IBDV标准强毒株BC6/85攻击.结果表明,胚胎免疫pCI-ChIL-18能显著促进试验鸡T、B淋巴细胞的早期增殖反应,增强IBDV多聚蛋白DNA疫苗诱导的早期抗体水平及其对强毒攻击的保护力.并进一步证明胚胎免疫pCI-ChIL-18对IBDV多聚蛋白DNA疫苗的早期免疫效果具有显著的增强作用(P相似文献   

肌注猪促生长激素释放因子(pGRF)基因质粒后猪神经内分泌生长轴激素的变化规律

试验探讨pGRF基因质粒在猪体内的表达效应及作用机理。将10头体重相近([15.24±0.53)kg]、遗传基础相似的健康杜×长×大三元杂交阉公猪安装颈静脉血导管,随机分成2组。试验开始时,试验组每头猪左侧臀部肌肉注射4.5mgpGRF基因质粒,第30d时,试验组每头猪右侧臀部肌肉注射相同剂量pGRF基因质粒,对照组两次分别注射2ml生理盐水。测定采样血浆中GRF、SS、GH及IGF-I的浓度。结果表明:①第一次注射pGRF基因质粒8d时,试验组GH的水平较对照组有显著提高(P<0.05),第12dGH水平达最高,为对照组的157%(P<0.01);第8、12、17、22d的IGF-I水平分别为对照组的185%(P<0.01)、237%(P<0.01)、200%(P<0.01)、131%(P<0.05);第12d试验组的SS水平较对照组有明显下降(P<0.05),其余时间无明显变化;GRF的水平在第12d分泌量达到最高,为对照组的143%(P<0.01),17d为对照组的133%(P<0.05),此后逐步下降又上升,与对照组的水平差异不显著。②在此试验条件下,基因质粒在5～8d开始缓释表达,12d左右达到表达高峰,其有效剂量持续时间在20d以上。③第二次注射基因质粒后,试验组第33d以后GH的水平逐渐升高,到45dGH水平达到峰值,为对照组的144%(P<0.05);第39、45、60d的IGF-I水平分别为对照组的166%(P<0.05)、173%(P<0.05)、117%(P<0.05);GRF的水平在45d分泌量达到最高,为对照组的138%(P<0.05),其余时间无明显变化;SS水平较对照组无显著差异。④相关分析表明:注射基因质粒后,试验组GH与GRF的相关系数为0.81(P<0.01),与SS的相关系数为-0.65(P>0.05);GH、GRF与IGF-I存在着正相关,SS与IGF-I存在负相关性,除GH与IGF-I的相关系数为0.67,达到显著正相关水平(P<0.05),其余指标与IGF-I均未有显著相关。     

Growth of ovine granulocyte-macrophage precursors in vitro without exogenous colony-stimulating activity

Ovine granulocyte-macrophage colony-forming units (CFU-GM) from peripheral blood and bone marrow were cultured in vitro. The colony-stimulating activity (CSA) was provided by various conditioned-media previously reported to contain CSA and by homologous sheep serum (SS). The maximum number of CFU-GM was observed in the cultures containing SS without the addition of exogenous CSA. The CFU-GM appeared earlier in the cultures containing bone marrow cells when compared to the peripheral blood CFU-GM. Replacement of SS by bovine fetal serum resulted in suboptimal growth of ovine CFU-GM.     

猪繁殖与呼吸综合征DNA重组质粒对猪的免疫效应

猪繁殖与呼吸综合征病毒(PRRSV)灭活疫苗、表达GP5蛋白的DNA重组质粒分别与表达IL-2和IL-4的重组质粒(pcDNA-IL-2和pcDNA-IL-4)联合免疫健康仔猪,经3次免疫后人工感染PRRSV HB-2株,检测仔猪体液免疫以及攻毒保护性反应。研究结果显示,重组质粒pCI-GP5可诱导免疫猪产生抗GP5抗体,最高ELISA抗体效价可达1∶285。攻毒后组织中PRRSV核酸的检出率下降30.3%,与对照组相比,差异显著(P<0.05),表明表达PRRSV GP5的DNA重组质粒pCI-GP5可诱导一定的免疫效力。pcDNA-IL-2与pCI-GP5联合免疫后,病毒血症的出现频率减少38.9%,PRRSV阳性组织检出率下降28.8%,与对照组差异显著(P<0.05);pcDNA-IL-4与pCI-GP5联合免疫后,最高ELISA抗体效价可达1∶320,病毒血症的出现频率下降38.9%,PRRSV阳性组织检出率减少34.8%,与对照组相比差异极显著(P<0.01)。本研究表明,PRRS DNA重组质粒pCI-GP5对猪的免疫保护力是稳定的,真核表达的细胞因子pcDNA-IL-2与pcDNA-IL-4能够显著增强pCI-GP5的免疫保护力。     

Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.     

Effect of long-term administration of porcine growth hormone-releasing factor and(or) thyrotropin-releasing factor on growth hormone, prolactin and thyroxine concentrations in growing pigs

Long-term administration of porcine growth hormone-releasing factor (pGRF(1-29)NH2) and(or) thyrotropin-releasing factor (TRF) was evaluated on serum concentrations of growth hormone (GH) thyroxine (T4) and prolactin (PRL). Twenty-four 12-wk-old female Yorkshire-Landrace pigs were injected at 1000 and 1600 for 12 wk with either saline, pGRF (15 micrograms/kg), TRF (6 micrograms/kg) or pGRF + TRF using a 2 x 2 factorial design. Blood samples were collected on d 1, 29, 57 and 85 of treatment from 0400 to 2200. Areas under the GH, T4 and PRL curves (AUC) for the 6 h (0400 to 1000) prior to injection were subtracted from the postinjection periods (1000 to 1600, 1600 to 2200) to calculate the net hormonal response. The AUC of GH for the first 6 h decreased similarly (P less than .05) with age for all treatments. The GH response to GRF remained unchanged (P greater than .10) across age. TRF alone did not stimulate (P less than .05) GH release but acted in synergy with GRF to increase (P less than .05) GH release. TRF stimulated (P less than .001) the net response of T4 on all sampling days. Animals treated with the combination of GRF + TRF showed a decreased T4 AUC during the first 6 h on the last three sampling days. Basal PRL decreased (P less than .05) with age. Over the four sampling days, animals injected with TRF alone showed (P less than .01) a reduction (linear effect; P less than .01) followed by an increase (quadratic effect; P less than .05) in total PRL concentration after injection; however, when GRF was combined with TRF, such effects were not observed (P greater than .10). Results showed that 1) chronic injections of GRF for 12 wk sustained GH concentration, 2) TRF and GRF acted synergistically to elevate GH AUC, 3) TRF increased T4 concentrations throughout the 12-wk treatment period, 4) chronic TRF treatment decreased the basal PRL concentration and 5) chronic GRF + TRF treatment decreased the basal concentration of T4.     

Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with 32P-labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72 h in culture) (P < 0.05, n = 4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144 h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P < 0.05, n = 4) and approximately seven-fold as compared to the 120-h cultures (P < 0.05, n = 4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120 h, but at 144 h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72 h) (P < 0.05, n = 4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells.     

Effect of porcine somatotropin on in vivo glucose kinetics and lipogenesis in growing pigs.

Crossbred barrows were used for in vivo studies investigating hormonal regulation of lipogenesis. The first experiment examined an in vivo method for determining rates of lipogenesis. Three barrows were infused with [U-14C]glucose and incorporation of radioactivity into triglycerides was determined in up to five biopsies of subcutaneous adipose tissue obtained over 7 h. Incorporation was linear after blood glucose specific radioactivity had reached a plateau and was constant over the entire infusion. For the second experiment, eight pigs (71 +/- 2.5 kg) were allocated to one of two treatments involving daily injections of excipient (control) or porcine somatotropin (pST; 120 micrograms/kg of BW). On d 10, beginning 15 h after injection, glucose incorporation into adipose tissue lipid was determined under both basal and hyperinsulinemic/euglycemic conditions. Basal glucose incorporation into lipid, particularly fatty acids, was markedly reduced (greater than 90%) during pST treatment. Although glucose incorporation was increased to a similar extent in both groups by hyperinsulinemia, the pST-treated pigs still exhibited markedly lower rates. Based on kinetic data, the decrease in lipid accretion of pST-treated pigs was primarily the result of a decrease in the rate of de novo synthesis. Furthermore, the reductions in glucose incorporation into fatty acids, glucose irreversible loss rate, and feed intake that occur with pST treatment were quantitatively similar.     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

Effect of bovine granulocyte colony-stimulating factor on the development of pneumonia caused by Mannheimia haemolytica

The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that the neutrophilia induced by recombinant bovine granulocyte colony-stimulating factor (GCSF) would lead to rapid bacterial clearance and less severe lesions after infection with M. haemolytica. Two experiments (A and B) were conducted in which four calves per experiment were treated daily with 5 microg/kg GCSF and four calves per experiment were treated with saline. All 16 calves were challenged with 5 x 10(9) colony-forming units (cfu)/ml (experiment A) or 4.5 x 10(8) cfu/ml (experiment B) of M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The mean maximal blood neutrophil counts in non-GCSF-treated and GCSF-treated calves before bacterial challenge were 5.6 +/- 0.7 x 10(9)/liter and 25.4 +/- 2.7 x 10(9)/liter, respectively. Two untreated calves became neutropenic and were euthanatized 2 days after infection because of severe respiratory distress. GCSF-treated calves had a 37% reduction in lung lesions compared with nontreated calves, and this difference was significant (P=0.04) when the effect of previous antibody titre to leukotoxin was considered. The effect of GCSF treatment on the severity of clinical signs seemed to be influenced by the antibody titre to M. haemolytica leukotoxin, although this effect could not be conclusively addressed. In conclusion, GCSF induced neutrophilia and partially protected calves against experimental infection with M. haemolytica. These results imply that increased numbers of neutrophils may, under some circumstances, protect against severe pneumonia caused by M. haemolytica.     

Effect of exogenous porcine somatotropin administration on nitrogen and energy metabolism in three genotypes of pigs

Effects of exogenous administration of porcine recombinant somatotropin (rpST) on protein gain and metabolic rate were measured in three genotypes (castrated males) of pigs (Pietrain, Duroc and a crossbreed between Dutch Yorkshire and Dutch Landrace). Six pigs of each genotype were assigned at approximately 60 kg to receive pST doses of either 0 (C) or 14 mg (T) administered i.m. twice weekly over 10 wk. Pigs were housed in individual metabolism cages at a room temperature of 20 to 22 degrees C and received feed at 2.6 times maintenance. Protein gain (N x 6.25) was measured over the final 6 wk of the 10-wk test period. For 2 wk in the test period (wk 2 and wk 5), six pigs of each treatment x genotype group were placed in a large respiration chamber and energy balances (in protein and fat) and metabolic rate were measured. Rate of weight gain measured over the final 6 wk of the experimental period increased by 105 g/d (13%) with rpST administration (P less than .01). Daily protein gain over 6 wk was increased by 48 g/animal with application of rpST (P less than .001). There was a genotype x treatment interaction (P less than .01) for protein gain. Daily protein gain in Durocs with pST treatment was increased (39%) more than in crossbreds (31%). Daily fat gain was decreased by 42 g/animal (P less than .001) by T. Daily heat production with rpST was increased by 12 kcal/kg.75, which is comparable to a 12% increase in the maintenance energy requirement.