Evaluation of elutriation and magnetic microbead purification of canine monocytes

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.     

Enrichment of T lymphocytes from bovine peripheral blood mononuclear cells using an immuno-affinity depletion technique ("panning")

A simple and efficient method to enrich bovine T lymphocytes from peripheral blood mononuclear cells (PBMC) by immuno-affinity depletion ("panning") has been developed. The PBMC were initially separated by density gradient centrifugation on Histopaque of density 1.077 g/ml. The T lymphocyte subset was then separated from PBMC by depletion of membrane immunoglobulin (Ig) bearing cells which had an affinity for anti-Ig antibodies bound to polystyrene tissue culture flasks. An average of 95% of the nonadherent "panned" cells were identified as T lymphocytes using a label of peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC). Two percent of the PNA negative cells were Ig bearing cells. The average yield was 50% of the original T lymphocytes found in the PBMC population, and the cell viability as assessed by trypan blue exclusion was greater than 95%. The separation took approximately 2 hours, and the total number of T lymphocytes recovered from 40 ml of blood was in the range of 20-40 X 10(6).     

Analysis of buoyant density of canine peripheral blood leukocytes with PVP-Silica (Percoll) density gradients

Isolation of canine peripheral blood mononuclear cells with a one step centrifugal separation procedure has not been very successful sofar. Significant contamination with polymorphonuclear cells has been reported. An analysis of the buoyant density of canine peripheral blood leukocytes on a self-generating Percoll gradient showed that the buoyant densities of polymorphonuclear cells and lymphocytes are so near that separation with high purity and yield is not possible with the use of a density gradient. Transient changes in buoyant density of polymorphonuclear cells have been observed. In such situations differences in buoyant density between cell types have been observed which permit separation of mononuclear cells from polymorphonuclear cells at a reasonable yield.     

In vitro properties of diffuse cytoplasmic esterase-positive canine mononuclear leukocytes

Depletion of cytoplasmic esterase-positive canine peripheral blood monocytes from mononuclear cell suspensions was attempted using plastic adherence, carbonyl iron ingestion and/or Sephadex G-10 filtration. An esterase-positive, nonadherent, nonphagocytic subpopulation was identified and further characterized by the presence or absence of cell membrane receptors for the Fc portions of immunoglobulin and the activated third component of complement. The majority of these nonadherent cells lacked these receptors. The data suggests that canine peripheral blood monocytes are a heterogenous cell population.     

Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.     

An improved method for the isolation of enriched canine peripheral blood mononuclear cell and peripheral blood lymphocyte preparations

A two-step purification method was developed for obtaining (1) peripheral blood mononuclear cell preparations of greater than 97% purity and (2) peripheral blood lymphocyte preparations of greater than 95% purity from canine whole blood with yields similar to or greater than those obtained by conventional techniques.     

The development of methods for immunophenotypic and lymphocyte function analyzes for assessment of Southern sea otter (Enhydra lutris nereis) health

The Southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act. The population began a pattern of slow decline in 1995. The decline was attributed to high adult mortality rates with infectious disease being the major cause of death. Multiple pathogens were implicated in these deaths including opportunistic pathogens such as Coccidiodes immitis and Toxoplasma sp. These findings suggested that the immunological health of mature animals in this population might be compromised. The primary goal of this study was to establish techniques for assessing phenotypic and functional baseline data for peripheral blood mononuclear cells (PBMC) in free-ranging sea otters. Standard total and differential white blood cell counts were augmented by emumeration of T and B lymphocyte subsets. Lymphocyte function was determined by both mitogen-induced proliferation and expression of IL-2 receptors. In addition to establishing normal ranges for adult animals, age-related changes were identified in B lymphocyte numbers and cell-surface density of major histocompatability complex class II (MHC II) proteins. The predominant lymphocyte subpopulation in Southern sea otters is the T lymphocyte. Substantial variation among individual animals was observed within the B lymphocyte population both in cell number and density of MHC II expression. Pups had greater numbers of T and B lymphocyte, as well as, greater MHC II expression on B lymphocytes than adults. Mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) was variable among individual animals with no significant difference in cell response between age class and gender. Concanavalin (ConA) was a more effective mitogen in stimulating proliferation and interleukin (IL)-2 receptor expression than pokeweed. This data can be used to augment routine hematology profiles and aid in the identification of animals with immunologic perturbations.     

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

猪外周血T淋巴细胞增殖反应MTT检测方法的建立

T细胞增殖反应是宿主T细胞识别病原的结果,也是宿主细胞免疫应答的重要指标之一。为了便于检测猪群在病原感染或者疫苗免疫过程中产生的细胞免疫应答,本研究应用MTT法建立了体外检测猪外周血T细胞增殖反应的研究方法。通过密度梯度离心法从外周血分离得到外周血单个核细胞（PBMC）,然后利用单核细胞和淋巴细胞不同的生长特性（贴壁与否）,弃掉贴壁的单核细胞,获得外周血淋巴细胞(PBL)。外周血淋巴细胞的流式分析结果显示,分离获得的PBL中T细胞所占比例达到了80%以上。应用MTT法分析了非特异性刺激物刀豆蛋白A（ConA）的浓度和细胞培养密度对T细胞增殖的影响。结果显示,ConA的工作浓度为5 μg/mL、细胞培养密度为2×106/mL时T细胞的增殖反应最强烈。本研究所建立的猪外周血T细胞增殖反应检测法可以为研究猪针对病原或疫苗的细胞免疫反应提供参考。     

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Cell surface markers of the canine natural killer (NK) cell

Studies were performed to determine which of several cell surface markers are expressed on canine peripheral blood leukocyte (PBL) natural killer (NK) cells. Chromium-51 release assays showed a decrease in NK activity after depletion of PBL by carbonyl iron ingestion and adherence to IgG-antibody-coated ovine erythrocytes (EA gamma) and to IgM-antibody-complement-coated ovine erythrocytes (EA mu C). Effector cell adherence to and subsequent lysis of canine thyroid adenocarcinoma (CTAC) target cell monolayers provided direct visual identification of the putative canine NK cell. These surface immunoglobulin-negative cells, individually identified by their physical adherence to dead CTAC target cells, failed to form nonimmune rosettes with guinea pig erythrocytes or rosettes with EA mu or EA mu C. However, 39.0 +/- 4.2% of these adherent cells formed rosettes with EA gamma and 73.3 +/- 0.8% expressed the canine T-lymphocyte marker, Thy-1.     

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

Activation specificity of commonly employed mitogens for canine B- and T-lymphocytes

Six day in vitro cultures of canine mononuclear leukocytes with or without prior carbonyl iron depletion were established with 5 commonly employed mitogens. Cultures were assessed for B-cell differentiation by induction of cytoplasmic and supernatant released IgG, M and A and T-cell differentiation by expression of Thy-1 antigen on the cell surface of lymphoblasts. The mitogen concanavalin A was shown to be a restricted T-cell mitogen, whereas pokeweed mitogen and protein A from Staphylococcus aureus stimulated maximal B-cell differentiation. These data establish the mitogenic specificity for canine lymphocytes in unfractionated peripheral blood leukocyte cultures and will permit an in vitro evaluation of various substances upon T- and B-lymphocyte functions.     

Murine monoclonal antibodies cross-reactive with feline peripheral blood mononuclear cells

Twelve murine monoclonal antibodies (MAB), specific for canine, human, and mouse cell surface determinants on lymphohemopoietic cells, were tested for reactivity (using indirect immunofluorescence) with peripheral blood mononuclear cells (PBMC) from 3 normal cats and 3 FeLV-positive cats with lymphoblastic leukemia. MAB DLy6 and 1H3, specific for canine lymphocytes, MAB HB57S, specific for human mu(IgM) chain, MAB J-118 40.164.3 and H81.98.71, specific for murine and human Ia, all had higher binding levels with PBMC from FeLV-positive cats compared to normal cats. Two MAB (S-78, S-24), specific for human class I determinants, cross-reacted with PBMC from both groups of cats. This panel of MAB may be useful for characterizing immunologically reactive cell subsets in normal as well as retrovirus-diseased animals.     

High Mobility Group Box 1-Protein expression in canine haematopoietic cells and influence on canine peripheral blood mononuclear cell proliferative activity

High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in mammalian tissues. HMGB1 demonstrates cytokine function and induces inflammation when actively released by haematopoietic cells or passively released during cell necrosis. This study aimed at the determination of HMGB1 expression in different cell types and at the evaluation of the role of HMGB1 in PBMC proliferation. Therefore we investigated the HMGB1 mRNA expression level in different canine haematopoietic cell types and the influence of exogenous rhHMGB1 on canine PBMC proliferation. Differentiated haematopoietic blood cells showed lower relative HMGB1 expression levels compared to CD34+ haematopoietic stem cells. Relative HMGB1 expression seemed also to decrease during differentiation of CD34+ stem cells into dendritic cells. Furthermore, peripheral blood CD14+ monocytes and granulocytes showed a lower relative HMGB1 expression in comparison to CD3+ T-lymphocytes. When exogenous rhHMGB1 at low concentrations was added to single PBMC cultures an increase of proliferation was obvious. However, in higher concentrations HMGB1 lost its stimulative effect. In conclusion, HMGB1 is broadly expressed in canine haematopoietic cells with highest levels in haematopoietic stem cells. HMGB1 induced directly PBMC proliferation.     

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures.The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 × 10⁵ cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 μCi tritiated thymidine (³H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.     

Natural killer (NK) activity and interferon (IFN) production by a fraction of spleen and blood lymphocytes in swine

After carbonyl iron treatment and gradient isolation, spleen and blood pig lymphocytes exhibited NK activity and produced IFN after viral induction. Removal of plastic-adherent cells, including the majority of B cells, did not change these activities. The plastic-non-adherent cells were further separated into two subsets of roughly similar size by panning using a monoclonal, anti-T, and anti-null cell antibodies (81 + cells). NK activity and IFN production were found in the 81 - cell fraction. A significantly higher proportion of null lymphocytes from blood and of splenic Fc-gamma receptor-bearing lymphocytes was also found among the 81 - cell fraction as compared to the 81 + fraction, without any change among other subsets. Similar proportions of helper (PT4+), cytotoxic (PT8+) and total T cells (MSA4+) were found among lymphocytes bound to target K562 cells and among the whole lymphocyte population. In contrast, lymphocytes that bound K562 cells demonstrated a striking increase in the proportion of Fc-gamma receptor-positive cells of high affinity. These results show that NK cells and IFN-producing cells are mainly included in the same blood and spleen fraction, and suggest that among 81 - cells only those expressing an Fc-gamma receptor of high affinity are active.