Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

Functional characterization of a swine CD4(+)/CD8(+) double positive lymphoblastoid T-cell line with a CD25(+)/CD45RA(-) phenotype generated in vitro with interleukin-2

A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2.     

Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

In vitro interactions of cytokines and bovine viral diarrhea virus in phytohemagglutinin-stimulated bovine mononuclear cells

Bovine viral diarrhea (BVD) virus inhibited phytohemagglutinin (PHA)-stimulated bovine peripheral blood mononuclear cell (PBMC) proliferation and bovine interleukin-2 (IL-2) production. In the controls, the heat-inactivated BVD virus was not capable of suppressing the PHA-stimulated PBMC proliferation. Presence of exogenous cytokines, such as purified human IL-2, recombinant bovine interleukin-1 (rbovIL-1), recombinant bovine IL-2, and recombinant human IL-6 failed to reverse the BVD virus-induced immunosuppression. Also, we found that the BVD virus inhibited PHA and IL-2 induced proliferation of bovine PBMC in the early and late stages of activation. In summary, our data suggest that BVD virus induced immunosuppression was not due to destruction of the PBMC but may be inhibiting one or more of the important intracellular enzymes that may regulate PBMC proliferation.     

Localization of interleukin-6 receptor mRNA in the pregnant and non-pregnant mouse uterus

To understand roles of interleukin 6 (IL-6) family cytokines for pregnancy in mice, localization of IL-6 receptor (IL-6R) mRNA was investigated in non- and early pregnant uteri by in situ hybridization. IL-6R mRNA was expressed in all non-pregnant uteri and in pregnant uteri from the third day (Day 3) to the sixth day of pregnancy (Day 6; the day of plug = Day 1). IL-6R mRNA signals were detected in non-pregnant mice in the luminal and glandular epithelium. Signal strength varied according to the sexual cycle. There was no correlation between the signal strength of the IL-6R mRNA and the serum concentrations of progesterone and 17beta-estradiol, which show a monophasic rise in the non-pregnant sexual cycle. In pregnant mice, slight signals were detectable in the luminal and glandular epithelium on Day 3. IL-6R mRNA messages increased with progression towards Day 4, however, localization changed drastically on Day 5. Stromal cells abruptly expressed their mRNA on Day 5, and these cells strongly expressed it on Day 6. The function of IL-6R in the luminal and glandular epithelium might be different from that in the stroma during the implantation period. In addition, few signals were identified in the stromal cells adjacent to the luminal epithelium on Day 6. This suggests that there are two types of stromal cells on Day 6 in mice.     

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.     

A monoclonal antibody to equine interleukin-4

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4⁺ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.     

Evaluation of serum interleukin 2 receptor and beta-2-microglobulin as prognostic factors for canine lymphoma: A pilot study

Interleukin 2 receptor (IL-2R) is released from activated T cell lymphocytes and related to proliferation of B cells and T cells. Beta-2-microglobulin (B2M) is synthesized from all nucleated cells and constitutes a major histocompatibility complex class I antigen. In human medicine, high concentrations of these two factors have been found to be related to prognosis in aggressive non-Hodgkin's lymphoma. In this pilot study, we aimed to assess the correlation between the serum concentration of IL-2R and B2M and the diagnosis and prognosis of canine lymphoma. This study included 8 healthy dogs and 17 dogs with lymphoma. To measure the serum concentration of IL-2R and B2M, a commercial enzyme-linked immunosorbent assay was used. In dogs with lymphoma, IL-2R concentrations were significantly high at the time of diagnosis, but B2M concentrations were not. In relapsed dogs, both IL-2R and B2M concentrations were significantly higher than those in the control and chemotherapy response groups. When the serum concentrations of IL-2R and B2M during chemotherapy were monitored in four relapsed dogs, B2M levels were more closely related with relapse. This study demonstrated that serum IL-2R and B2M concentration can be a diagnostic or prognostic tool for canine lymphoma. Monitoring of serum B2M concentration seems to be useful for predicting relapse.     

Molecular characterization of canine BCR‐ABL–positive chronic myelomonocytic leukemia before and after chemotherapy

Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High‐resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region‐Abelson murine leukemia viral oncogene homolog (BCR‐ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR‐ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.     

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (Fc_γRIII and Fc_γRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block Fc_γRs and has been used to treat a variety of canine autoimmune disorders. Fc_γRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage Fc_γR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate Fc_γR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fc_γ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.     

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.     

Production and characterization of monoclonal antibodies reactive with the chicken interleukin-15 receptor alpha chain

We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor.     

H3亚型犬流感病毒的荧光定量PCR方法建立及其增殖特性研究

旨在建立H3亚型犬流感病毒(canine influenza virus,CIV)荧光定量PCR方法,利用该方法对病毒在犬肾细胞(MDCK细胞)上增殖特性进行研究.根据CIV的血凝素(HA)片段上的HA2基因保守区设计并合成特异性引物,构建CIV-HA重组质粒作为标准品,经过条件优化,建立H3亚型CIV荧光定量检测方法...     

稳定表达犬细小病毒VP2结构蛋白的CHO-K1细胞系的建立

为构建犬细小病毒VP2基因分泌性表达细胞系,通过酶切将人CD5信号肽序列从质粒中切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法扩增犬细小病毒VP2基因,并将其插入到pcDNA3.1-CD5sp载体中CD5信号肽的下游,使其与CD5信号肽序列融合,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经脂质体介导转染细胞,后通过G418筛选,建立出稳定表达VP2蛋白的CHO-K1细胞系。测序结果表明,构建的犬细小病毒VP2基因的分泌型表达载体结构正确,表达载体经脂质体介导转染CHO-K1细胞,通过G418加压,筛选出稳定转染VP2基因的细胞株,经PCR检测证明VP2基因已经整合到细胞的染色体中;经RT-PCR、Westernblot分别检测VP2基因表达的mRNA和VP2蛋白,证明犬细小病毒VP2基因能够在CHO-K1细胞进行稳定性表达。这为下一步研究犬细小病毒VP2蛋白与宿主细胞的相互作用及VP2DNA疫苗奠定了基础。