Cloning of full length cDNA, high-level expression and biological characterization of feline IL-12

A full length cDNA of feline interleukin(IL)-12 p35 and p40 subunits was cloned. By transferring the plasmids containing both the subunit genes to mammalian cells, we expressed biologically active feline IL-12. The expressed feline IL-12 has interferon-gamma-inducing activity against both human and feline peripheral blood mononuclear cells (PBMC) and stimulates cytotoxic T lymphocyte activity against herpes simplex virus-infected human PBMCs. There were two kinds of molecules (p75, p80) in the purified recombinant feline IL-12, and both molecules exhibited biological activity. The difference between p75 and p80 was the degree of the glycosylation of the p35 chain. Moreover, when we modified the cDNA of p35 by changing some codons and deleted the 5' and 3' non-coding regions, the expression level of IL-12 increased about 100 fold.     

Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.     

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease.     

Towards non-invasive imaging of HSV-1 vector-mediated gene expression by positron emission tomography

The overall goals of the broad and growing field of molecular medicine is to identify fundamental errors of disease and to develop corrections of them on the molecular level. At the same time, real-time imaging of gene expression in vivo aims towards a detailed analysis of both endogenous and exogenous gene expression in animal models of disease and in the clinical setting. Non-invasive imaging of endogenous gene expression may reveal insight into the molecular basis of disease pathogenesis and the extent of treatment response. When exogenous genes are introduced, e.g. by herpes simplex virus type 1 (HSV-1)-based vectors, to ameliorate a genetic defect or to add an additional gene function to cells, imaging techniques may reveal the assessment of the location, magnitude and duration of therapeutic gene expression and its correlation to the therapeutic effect. Here, we review the main approaches of non-invasive imaging techniques of gene expression in vivo with special reference to HSV-1 vector-mediated gene expression.     

Bioactivity and secretion of interleukin-18 (IL-18) generated by equine and feline IL-18 expression constructs

Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bioassay for human IL-18 was recently described using human myelomonocytic KG-1 cells, which produce human IFNgamma in response to human IL-18 in a dose dependent manner (Konishi et al., 1997). We demonstrated bioactivity of equine and feline IL-18 protein in transfection products of CHO cells using this assay. Bioactivity of ILRAP-IL-18 protein was demonstrated in the culture medium of transfected CHO cells. These data imply that the ILRAP-IL-18 construct shows potential for use in vivo, where cell secretion of protein is crucial.     

PRRSV变异株ORF6基因与IL-18共表达真核质粒的构建及其表达

针对白细胞介素18(IL-18)基因和本实验室分离的PRRSVJL/07/SW毒株全基因序列,分别设计了2对特异性引物,扩增出IL-18基因和ORF6基因。将2个目的基因克隆于真核表达载体pEGFP-N1中,成功构建了重组真核质粒pEGFP-ORF6和pEGFP-IL18-ORF6,阳性质粒转染Marc-145细胞进行筛选。通过表达载体在转染细胞中的高效转染,增强型绿色荧光蛋白(EGFP)快速、准确的反映出阳性质粒在细胞质中的正确表达,再通过SDS-PAGE和Western blot鉴定构建的真核表达质粒pEGFP-IL18-ORF6的正确性。结果表明,ORF6基因和IL-18基因在Macr-145细胞中能有效表达。结论,所构建的真核质粒pEGFP-IL18-ORF6结构正确,能够在Marc-145细胞中高效表达,而且表达产物具有特异免疫学反应。     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.     

The feline acute phase reaction

The acute phase reaction (APR) is a response to potentially pathogenic stimuli. It begins with the release of interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha from inflammatory cells. These cytokines induce fever, leucocytosis and release of serum acute phase proteins (APPs). In this review, the characteristics of the feline APR are described. In cats with inflammatory conditions, fever is a common finding, with leucocytosis due to the release of cells from the marginal pool, followed by activation of myelopoiesis. Because excitement frequently causes leucocytosis in cats, a diagnosis of inflammation should therefore be supported by additional findings such as the presence of toxic neutrophils. The major APPs are serum amyloid A and alpha(1)-acid glycoprotein (AGP), which both increase a few hours after the inflammatory stimulus and remain elevated for as long as the inflammation persists. AGP plays an important role in the diagnosis of feline infectious peritonitis (FIP) and may also be useful also in studies of FIP pathogenesis.     

Interleukin 1 alpha mRNA-expressing cells on the local inflammatory response in feline infectious peritonitis.

By in situ hybridization with biotinylated human interleukin 1 alpha (IL-1 alpha) cDNA probe, distribution of feline IL-1 alpha mRNA-expressing cells was examined in the tissues from 49 cases diagnosed as feline infectious peritonitis (FIP) by pathological examination. IL-1 alpha mRNA-expressing cells were found in visceral peritoneum, lymphoid organs, liver, kidney, pancreas, digestive tract, lung, pleura, brain, palpebral conjunctiva, and bone marrow. Hybridization signals for IL-1 alpha mRNA were mostly located in the local inflammatory lesions on the serosal surface of various organs and omentum, which were frequently involved in the lesions of FIP (27.8 +/- 5.1 cells/mm2). Morphological examination suggested that they were infiltrated macrophages. However, few IL-1 alpha mRNA-expressing macrophages were in the lesions of other organs. These data suggested that IL-1 alpha produced from macrophages in the local inflammatory sites might participate in the initiation and development of the lesions on the visceral peritoneum in FIP.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

Parameters of production and partial characterization of feline interleukin 2

The conditions for the production of feline interleukin 2 (IL-2) from peripheral blood leukocytes (PBL) and splenocytes by concanavalin A (Con A) stimulation are described. Feline IL-2 was quantitated by measuring DNA synthesis in the murine IL-2-dependent cell line, CTLL-20. In addition, feline IL-2 was generated for the maintenance of long-term cultures of Con A-stimulated feline PBL and for biochemical characterization. Finally, IL-2 production was evaluated from the PBL of feline leukemia virus (FeLV)-infected cats. Con A at 9.6 micrograms/ml produced a plateau of peak IL-2 activity from 24 to 48 h following stimulation. The tumor promoter, phorbol myristic acetate, stimulated feline IL-2 production and enhanced Con A-stimulated feline IL-2 production. Fetal calf serum (FCS) was not required for IL-2 production; however, FCS at 5% (v/v) allowed for maximal Con A-stimulated IL-2 production. Feline IL-2 generated from Con A-stimulated splenocytes migrated with an apparent molecular size of 13.7 to 23 kD by gel filtration chromatography and supported the proliferation of Con A-activated feline PBL at a final concentration of 0.3 to 0.9 units/ml.     

In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.

In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.     

β防御素124沉默通过p38MAPK/AP1通路调控山羊附睾头细胞趋化因子和细胞因子的表达

旨在研究山羊β防御素124（goat beta-defensin 124,gBD124）沉默对p38MAPK/AP1通路及下游细胞趋化因子和细胞因子表达的影响。本研究将设计的3条gBD124-shRNAs载入LV10-U6/RFP&Puro质粒载体,与包装质粒共转染到293T细胞中,用梯度稀释法测定病毒原液的滴度;将构建的3个LV10-gBD124和LV10-gBD124-NC载体转染到山羊附睾头细胞中,筛选有效沉默载体;然后用有效沉默载体联合转染附睾头细胞,设置了空白细胞对照组、LV10-NC组、有效沉默载体组,用2 μg·mL^-1嘌呤霉素筛选后分别收集细胞和培养液,采用qRT-PCR、Western blot和ELISA检测gBD124、MAPK信号通路关键蛋白、细胞因子及趋化因子基因及蛋白表达情况。本研究构建了gBD124沉默慢病毒载体,筛选出LV10-gBD124-51和LV10-gBD124-161两个有效载体,并成功构建了gBD124沉默稳定转染的附睾头细胞株。与NC对照组相比,gBD124沉默显著降低了山羊附睾头细胞MAPK通路中MAPK1以及AP1的两个亚型c-JUN和c-FOS基因表达（P<0.05）,显著增加了RASA1基因表达（P<0.05）;gBD124沉默显著降低了总p38MAPK、总c-JUN和总c-FOS蛋白表达,以及磷酸化p38MAPK和c-JUN蛋白表达（P<0.05）;gBD124沉默显著上调了MAPK通路下游IL-1β及其受体IL-1R2、IL-8和趋化因子CCL6和CCL21基因表达（P<0.05）,下调了CCL5和IL-1α基因表达（P<0.05）;gBD124沉默显著降低了细胞培养液中CCL5浓度（P<0.05）。与空白对照组相比,gBD124沉默显著增加了培养液中IL-1β和IL-8浓度,降低了IL-1α浓度（P<0.05）。gBD124基因沉默通过抑制p38MAPK/AP1信号通路调控附睾头细胞趋化因子和细胞因子的表达。     

Development of an in vitro model of feline cartilage degradation

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1α, or IL-1β. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1β and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1β and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1β dose-response studies showed that an IL-1β dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1β did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.