The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs

A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.     

Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the 3-day culture period. To establish basal conditions hepatocytes were maintained in serum-free William's E medium containing 10 nM dexamethasone and 1 ng/ml insulin. For the final 24 h, insulin (1 or 100 ng/ml) or glucagon (100 ng/ml), were added in the presence or absence of 100 nM triiodothyronine (T3). RNA was extracted and quantitative RT-PCR was performed with primers specific for the long form and total porcine leptin receptors. Leptin receptor expression was calculated relative to co-amplified 18S rRNA. Expression of the long form of the leptin receptor was confirmed under basal conditions. Insulin, glucagon and synthetic human proteins (ghrelin and GLP-1) at 100 ng/ml had no influence on leptin receptor expression; the addition of T3 was associated with a marked increase (P < 0.001) in expression of total and long forms of the leptin receptor by 1.6 and 2.4-fold, respectively. Addition of leptin to cells which were pre-treated with T3 for 24 h (to up-regulate leptin receptor expression), confirmed the lack of a direct effect of leptin on glucagon-induced glycogen turnover and cAMP production. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3.     

大鼠原代肝脏细胞的分离培养及其膜受体鉴定

本研究采用改良的两步胶原酶灌流法分离培养大鼠肝脏细胞,并对其进行了形态学观察、功能检测及表面受体的鉴定,以探索其短期培养的最佳功能状态。结果表明,平均每只大鼠可获取2.12×10⁸个肝脏细胞,平均活率达94.23%;光镜下观察发现肝脏细胞呈多角形并成片生长;对肝脏细胞的LDH、Alb和尿素进行连续检测,结果发现细胞离体培养第3天功能较好;Mab263-PE单抗鉴定结果显示,肝脏细胞生长激素受体功能健全尚未受损,为细胞信号转导等相关试验奠定了基础。     

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).     

小鼠肝实质细胞的分离、鉴定及原代培养

为分离、培养高纯度原代小鼠肝实质细胞,并鉴定其纯度及生物活性,试验采用原位两步循环灌流法及多次低速差速离心法分离纯化肝实质细胞,促贴壁培养基原代培养,台盼蓝检测其存活率,PAS反应检测其糖原合成能力,免疫荧光化学检测细胞中CK18表达情况。结果表明:每只小鼠可获取约1.5×10^6个细胞,存活率>97%;镜下发现细胞在接种后6h开始贴壁,72h贴壁细胞生长状态良好,胞体变大、不规则,细胞间相互靠拢呈岛状或条索状连接;肝细胞出现成片紫红色糖原颗粒,CK18在细胞中均匀分布;糖原反应联合CK18免疫荧光显示细胞纯度在90%以上。说明该试验所用方法分离出肝实质细胞数量和存活率高,促贴壁培养基培养的肝实质细胞纯度高,为细胞代谢、细胞毒性等的研究奠定了基础。     

无血清培养关中奶山羊胚胎生殖细胞的研究

山羊胚胎生殖细胞是一种来源于胎儿原始性腺的多能干细胞,建立该细胞体外稳定分离培养体系对研究山羊繁殖育种具有重要价值。本试验通过酶消化法和组织培养法分离培养关中奶山羊胚胎生殖细胞,检测无血清培养基对细胞体外增殖的影响。结果发现,该培养基可以分离得到山羊胚胎生殖细胞,细胞集落形态典型,表达AKP、Oct4、TERT及SSEA-1。经体外分化试验表明,细胞可以分化为类胚体、成纤维样细胞、成脂细胞和卵母细胞样形态。无血清培养基可以用于山羊胚胎生殖细胞的分离与培养,本试验对进一步建立山羊胚胎生殖细胞长期培养体系提供了新的参考。     

Porcine preadipocyte proliferation and differentiation: a role for leptin?

The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig s.c. adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on six-well tissue culture plates and proliferated to confluency in 10% (vol/vol) fetal bovine serum (FBS) in Dulbecco's modified Eagle medium/F12 (DMEM/F12; 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol) and recombinant porcine leptin at concentrations of 0 to 1,000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 microM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL). The GPDH and LPL activities are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 h after seeding cells with 10% FBS in DMEM/F12 in 25-cm2 tissue culture flasks, cells were switched to 5% FBS and supplemented with 0 to 1,000 ng of porcine leptin or 1,000 ng of murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 h on d 4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1 microM dexamethasone) for 7 d. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone, or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL. Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone, or IGF-I; however, leptin at 1,000 ng/mL stimulated a 30% increase in preadipocyte proliferation (P = 0.007; n = 6) and a 27% increase in stromal cell proliferation (P < 0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.     

Sf9昆虫细胞悬浮培养工艺的研究

对Sf9昆虫细胞在不同培养基、培养基中是否添加血清及不同生物反应器中的培养工艺进行了研究,发现Sf9细胞在Sf900Ⅱ无血清培养基中生长比在添加10％小牛血清的Grace培养基中更好,在Sf900Ⅱ无血清培养基14 L搅拌式生物反应器分批培养细胞密度可达1.4×107/mL.过程生化特性分析表明,总氨基酸的消耗及主要代谢副产物特别是乳酸及游离氨的积累是培养后期细胞密度降低及活性下降的重要原因.本研究为Sf9细胞生物反应器培养工艺优化及利用昆虫杆状病毒蛋白表达系统高效表达重组蛋白生产亚单位疫苗奠定了良好基础.     

Hypothermic storage of equine isolated hepatocytes

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.     

In vitro growth-promoting activity of porcine mammary secretions: initial characterization and relationship to known peptide growth factors

We have examined the in vitro growth-promoting properties and growth factor content of porcine mammary secretions. Defatted, porcine colostrum stimulated the proliferation of fibroblast and epithelial-like cell lines of diverse species origins in serum-free medium and cellular DNA synthesis (4- to 119-fold) as monitored by uptake of 3H-thymidine into DNA of quiescent cells in culture. Porcine milk, although mitogenic, had reduced activity when compared with colostrum on an equivalent-volume basis. Furthermore, the relative mitogenic activity of milk, although still detectable at 3 wk, continued to decline with length of the lactation period. Fractionation of pig colostrum on gel-filtration columns revealed multiple peaks of (AKR-2B) fibroblast mitogenic activity (208, 66 and 4.6 kdaltons) and a heterogenous profile of epithelial cell mitogenicity. Polyclonal antibodies (IgG) specific for murine epidermal growth factor (EGF; the major mitogen in human and murine milk) or human platelet-derived growth factor (PDGF) did not inhibit the mitogenic activity of pig colostrum or milk, demonstrating lack of antigenic relatedness between the contributing porcine factors and mEGF or hPDGF. Also, we were unable to demonstrate similarity of the small Mr colostral factor with EGF by use of EGF radioreceptor assay. These results identify porcine colostrum and milk as sources of potentially important in vitro growth-promoting factors. The enhanced expression of these factors in early mammary secretions suggests their possible in vivo involvement in mammary and neonatal tissue growth processes.     

Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles. One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age). Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells. Muscles were digested after the removal of all visible connective tissue from the excised muscle. One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein. The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6. Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures. Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures. The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures. Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures. These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes.     

Epidermal Tissue-Derived T-cell Growth Factor,Colony Stimulating Factor and Nerve Growth Factor in Chickens

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9- to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).     

Expression Patterns of T1R1 and T1R3 in the Congjiang Xiang Pig during Epididymal Development

The aim of this experiment was to study the expression pattern of taste receptor family 1 subtypes 1 (T1R1) and 3 (T1R3) during epididymal development of Congjiang Xiang pig, and to explore the possible role of these taste receptors in mammalian male reproductive function and its potential medical value. In this study, the differential expressions of T1R1 and T1R3 in epididymis at 4 key developmental periods (neonatal (15 d), peri-puberty (30 d), puberty (60 d) and sexual maturity (180 d)) of Congjiang Xiang pigs were analyzed. RT-qPCR, immunohistochemistry (IHC) and Western blot were used to detect the changes and distribution of the two taste receptors in epididymis of Congjiang Xiang pigs at different ages. The results of RT-qPCR showed that the expression of TAS1R1 and TAS1R3 mRNA increased gradually from neonatal (15 d) to sexual maturity (180 d), and there was a significant difference between each period (P<0.01). The results of Western blot showed that the expression of T1R1/T1R3 protein was the highest on the 180 d and the lowest on the 15 d. The average protein abundance of T1R1/T1R3 was as follows: 180 d > 30 d > 60 d > 15 d. The results of IHC showed that T1R1 and T1R3 proteins were distributed in the epididymis of Congjiang Xiang pigs at 4 periods, in which T1R1 protein was mainly concentrated in epithelial cell membrane, especially in basal and narrow cells, while T1R3 protein was strongly positive in stereocilia, annular vacuoles and spermatozoa. In summary, the expression of T1R1/T1R3 in the epididymis of Congjiang Xiang pigs increased gradually from 15 d to the peak of sexual maturation, which was related to the differential expression of T1R1/T1R3 in epithelial basal cells, narrow cells and stereocilia of epididymis. These special expression patterns were time related to the physiological function of epididymis, so it is speculated that T1R1/T1R3 are involved in the regulation of sperm maturation and storage in epididymis.     

T1R1和T1R3在从江香猪附睾发育中的表达模式

为研究味觉受体第一家族亚型1（T1R1）和3（T1R3）在从江香猪附睾发育过程中的表达模式,探讨味觉受体在哺乳动物雄性生殖机能中可能发挥的作用及潜在医学价值,本试验以从江香猪附睾组织为研究对象,分析附睾发育4个关键时期：初情前（15 d）、初情时（30 d）、初情后（60 d）和性成熟期（180 d）T1R1与T1R3的差异表达。采用实时荧光定量PCR、免疫组织化学（IHC）和Western blot检测两个味觉受体在不同日龄从江香猪附睾组织中转录、翻译水平的变化及其分布情况。RT-qPCR结果表明：TAS1R1与TAS1R3 mRNA在从江香猪附睾初情前（15 d）至性成熟期（180 d）表达量逐渐增加,且任意两个时期间差异极显著（P<0.01）。Western blot结果显示,T1R1/T1R3蛋白在180 d表达量最高,在15 d表达量最低,两者之间差异显著（P<0.05）,平均表达丰度依次为180 d > 30 d > 60 d > 15 d。IHC结果显示,T1R1和T1R3蛋白在各日龄组从江香猪附睾组织均有分布,其中T1R1蛋白主要在上皮细胞膜上,尤其是基细胞和窄细胞;而T1R3蛋白主要在微绒毛、环状空泡和精子呈强阳性表达。综上,本研究发现不同日龄从江香猪附睾的T1R1/T1R3表达从15 d逐渐增加,至性成熟达到峰值,这一表达变化与附睾上皮基细胞和窄细胞及微绒毛的T1R1/T1R3的差异表达有关,这些特殊的表达模式与附睾生理功能存在时间关联,故推测T1R1和T1R3参与附睾内精子成熟和储存的调节过程。     

Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.     

新生仔猪肝脏发育的肝细胞生化分析

选择新生仔猪15头，分别于出生当日(0 d)、出生后3d及7d屠宰取样，制作肝脏电镜切片进行组织学分析，并测定肝脏中DNA、RNA含量及常规生化指标。结果显示：新生仔猪肝细胞中内质网、线粒体等细胞器都很丰富，而且各细胞器的结构已经发育成熟。肝脏质量在仔猪出生后1周尤其是3d内迅速增加(P＜O．01)，远远快于体重增加的速度。仔猪肝脏的蛋白质含量在生后1周也迅速增加(P＜O．01)；而肝糖原贮备在刚出生时较高，出生后迅速下降(P＜O．01)。仔猪出生后肝脏中DNA和RNA浓度的变化趋势正好相反，前者在出生后3d内明显下降(P＜O．05)，4～7d又有增加趋势，而RNA浓度以3d为最高。试验结果表明，仔猪到出生时肝细胞的结构和功能基本发育成熟。仔猪出生以后，肝脏的生长发育十分迅速，不仅肝细胞的数量快速增加，肝细胞的体积也不断增大。     

Effects of surgical catheterization and degree of isolation on the behavior and exocrine pancreatic secretion of newly weaned pigs

The purpose of this study was to examine the effects of surgery and isolation on behavior and exocrine pancreatic secretion of nursery pigs. Thirty-two pigs were weaned at 28 d (d 0) of age and assigned to one of four treatments that comprised the 2 x 2 factorial combination of surgery (surgically modified, intensively handled animals or intact, minimally handled animals) and isolation (fully or partly isolated). The pigs were kept in metabolic crates that provided full or partial isolation for 25 d after weaning. On d 3, one-half of the fully and partly isolated pigs were surgically fitted with a pancreatic catheter, whereas others were left intact. Exocrine pancreatic secretions were collected from catheterized pigs beginning on d 7 and continuing every 2nd d. Behavior of the pigs was video-recorded on d 6 and 13 from 0700 to 2230. There was no indication of treatment effects on the exocrine pancreatic secretion. The volume of pancreatic secretion increased from d 5 to 23, and during the same period the output of protein and enzyme activity also increased. Surgically catheterized pigs showed a passive behavioral response in their undisturbed behavior, characterized by less time spent active, fewer postural changes, less chain manipulation, fewer escape attempts, and less play behavior than their intact littermates. Furthermore, surgical catheterization led to less fear of humans and behavioral indications of an ability to distinguish between familiar and unfamiliar persons. Partial isolation led to increased activity, increased chain manipulation, and increased play behavior, indicating that the provision of limited social contact may help reduce the negative effects of individual housing in weaned pigs. These results suggest that it is important to be aware of effects of housing, experimental surgery, and associated handling when interpreting results from intensive experiments.     

Regulation of DNA synthesis in Leydig cells obtained from neonatal pig testes.

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.     

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages

This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.     

Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation

The morphological and biochemical characterisation of adult chicken hepatocytes in a serum-free culture are described. When cultured in positively charged plastic dishes, chicken hepatocytes formed a monolayer cell sheet. The monolayer morphology of these chicken hepatocytes was quite distinct from the spheroid shape of rat hepatocytes cultured under similar conditions. Electron microscopy showed that the cytoplasmic organelles of chicken hepatocytes were well preserved in vitro. Two-dimensional gel electrophoresis showed that the chicken hepatocytes secreted liver-specific proteins. Several enzymes of glucose-6-phosphatase, cytochrome P-450 or glutathione S-transferase, involved in metabolic and biotransformation pathways in the liver, were retained in the chicken hepatocytes in a serum free condition. These findings suggest that the primary culture of adult chicken hepatocytes with a serum-free culture system could be useful to study the hepatic metabolic pathway in the chicken and its response to various chemicals.