Secretion of dopamine and norepinephrine in hypophyseal portal blood and prolactin in peripheral blood of Holstein cattle

The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle, and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated controls (SOC). Holstein heifers (606 +/- 21 kg BW; mean +/- SE) were subjected to neurosurgery for 8 h to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20-40 ng/ml throughout 255 min. PRL abruptly increased and remained above 90 ng/ml after HST compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 min. Within 5 min after severing the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P < 0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P < 0.05) than in peripheral blood during the first 60 min. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.     

A procedure for parallel isolation of white blood cells, granulocyte and purified neutrophil suspensions from the peripheral blood of cattle.

A rapid method is described for parallel isolation of white blood cells, granulocytes and purified neutrophils from peripheral blood of normal cattle. The mean recovery (+/- S.D.) of white blood cells, granulocytes and purified neutrophils isolated from peripheral blood of 13 cows was 66.4 +/- 12.6%, 68.7 +/- 20.0% and 38.0 +/- 20.9%, respectively. The mean purity of the isolated granulocyte and neutrophil suspensions was 94.0 +/- 3.8% and 95.0 +/- 6.0%, respectively. Viability of isolated cells was more than 97%.     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

A study of peripheral blood in hedgehogs in Turkey

The aim of this study was to determine diameters of blood cells, differential counts of peripheral blood leukocytes, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (ACP-ase) activity of some leukocyte types, and enzymatic positivity percentages of peripheral blood lymphocytes in two hedgehogs species, Hemiechinus auritus, the long-eared hedgehog, and Erinaceus concolor, the southern white-breasted hedgehog. Air-dried peripheral blood smears were stained with May-Grünwald-Giemsa stain. ANAE and ACP-ase were stained in glutaraldehyde-acetone-fixed smears. ANAE-positive lymphocytes displayed a dot-like positivity pattern characterized with 1-5 reddish brown cytoplasmic granules, whereas ACP-ase positive lymphocytes displayed a dot-like positivity pattern characterized with 1-3 pinkish cytoplasmic granules. Monocytes gave a diffuse and strong reaction while neutrophils displayed a weak positive reaction for ANAE and ACP-ase. No difference was observed in mean diameters of peripheral blood cells of these species. It was found that lymphocytes made up the majority (64.3% and 65.5%) of leukocytes, followed by neutrophils (23.9% and 23.3%), eosinophils (9.0% and 7.6%), monocytes (1.8% and 2.3%), and basophils (1.0% and 1.3%) in H. auritus and E. concolor, respectively. Mean ANAE positivity oflymphocytes was 36.6% and 51.3% and ACP-ase positivity was 32.1% and 37.5% for H. auritus and E. concolor, respectively. The ANAE positivity of lymphocytes in E. concolor was significantly (P < 0.05) higher than that of H. auritus.     

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.     

Canine dendritic cells from peripheral blood and lymph nodes

Canine dendritic cells were prepared from peripheral blood or lymph nodes using a series of steps including fractionation on bovine plasma albumin (BPA), irradiation with 4000 R, incubation for 16–18 hours, and refractionation on BPA. Dendritic cells were recovered in the low density (LD) fraction containing approximately 0.6% of the unfractionated cells. Measured by the incorporation of ³H-thymidine, the response of the high density (HD) cells to neuraminidase-galactose oxidase (NGO) was lower than that of the unfractionated lymph node cells (LNC) but increased in a concentration dependent manner after the addition of a population of cells enriched for dendritic cells (30–70% by morphologic criteria). Cooperation between HD- and LD- cells was not restricted to identity of the major histocompatibility complex. Canine dendritic cells also displayed stimulatory activity higher than unfractionated peripheral blood mononuclear cells (PBMC) in a one way mixed leukocyte culture (MLC). Canine dendritic cells were nonadherent to plastic, were of low density, and remained viable and functional after irradiation. For the first time, canine dendritic cells have been identified in peripheral blood and lymph nodes and have been shown to act as accessory cells in the response of lymphocytes to NGO and as stimulator cells in a MLC.     

Spontaneously active suppressive cells in canine peripheral blood

The phenomenon of the unusually high spontaneous suppressive activity of cells in peripheral blood of dogs was analysed. The m/c (mitomycin C)-treated population of peripheral blood leucocytes (PBL) contained cells able to reduce the responsiveness of autologous cells by 48 +/- 15% (P less than 0.01) and their activity was not indomethacin dependent. Thoracic duct lymphocytes (TDL) did not reduce the response of PBL to PHA, neither did cell crowding. The supernatants from 24-h cultures of m/c-treated PBL did not affect the response to PHA, and parallelly precultured cells inhibited the proliferation of PBL to a lesser degree (24 +/- 9%) than the fresh cells (50 +/- 16%, P less than 0.05). Addition of m/c-treated polymorphonuclear cells at PMN to PBL ratios of 1:4 and 1:1 progressively inhibited PBL reactivity to PHA, from 29.5 +/- 3.5% to 68.5 +/- 9%, respectively, and the supernatants from 24-h cultures of PMN reduced the proliferation by 48 +/- 2.8%. The neutrophil-derived inhibitory factor(s) was non-cytotoxic and reduced the formation of blasts to 61.5 +/- 3.5% of the control values. These results indicate that dog PBL from Lymphoprep gradient contain a population of non-recirculating, short-lived, spontaneously suppressive cells, mainly PMN, which modulate T cell reactivity in vitro, suggesting that neutrophils may be able to exert a regulatory effect in vivo.     

Analysis of ovine peripheral blood lymphocyte subsets following Ficoll-Hypaque separation or erythrocyte lysis

Peripheral blood lymphocyte subsets were analysed with the aid of monoclonal antibodies and flow microfluorometry following separation by either the Ficoll-Hypaque method or erythrocyte lysis of the buffy coat. The percentage of lymphocytes labelled by the monoclonal antibodies SBU-T1, SBU-T4 and SBU-T8 were significantly greater (P less than 0.05) in samples obtained by erythrocyte lysis. In contrast, a lower percentage of lymphocytes was labelled by SBU-T6 following erythrocyte lysis (P less than 0.02). These data suggest caution when choosing a method for separation of peripheral blood lymphocytes.     

Distribution of glutathione peroxidase activity and selenium in the blood of dairy cows.

The distribution of glutathione peroxidase (GSH-Px) activity and selenium concentrations among several components in the blood of dairy cows was examined. Approximately 98% of the GSH-Px activity in peripheral blood was associated with the erythrocytes when enzyme activity was expressed as units per milliliter of blood. The GSH-Px activity expressed per cell was approximately fourfold greater for peripheral leukocytes than for erythrocytes. The cellular component contained a greater proportion (approx 73%) of whole blood selenium than did the plasma. A positive linear relationship (r = 0.958) between blood GSH-Px activity and blood selenium concentrations was found in dairy cattle under practical field conditions.     

Whole blood leukocyte vs. separated mononuclear cell blastogenesis in calves: time-dependent changes after shipping.

The blastogenic response of peripheral blood mononuclear cells to mitogenic stimulation by concanavalin A was lower (P less than 0.01) after transporting 60 dairy calves 480 km than it was either one or two weeks later. The response was similar for phytohemagglutinin. There was a decrease (P less than 0.05) in the number of peripheral blood monocytes and neutrophils two weeks after shipping. The transportation of calves did not affect plasma IgG1 or IgM level. The mitogenic stimulation of peripheral blood leukocytes by both phytohemagglutinin and concanavalin A in whole blood cultures was more variable than with the culture of peripheral blood mononuclear cells. Technique variation, which was defined as the coefficient of variation among quadruplicate cultures, was greater than 20% for while blood assays and less than 10% for cultures of peripheral blood mononuclear cells. The variation among different calves tested at the same time and the variation within single calves tested at different times were also lower in peripheral blood mononuclear cell cultures than in whole blood mononuclear cell cultures than in whole blood assays. It is suggested that the variation among replicate cultures be reported in blastogenesis studies.     

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.     

Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: individual variability and repeatability

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.     

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).     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

CD56 is expressed exclusively on CD3+ T lymphocytes in canine peripheral blood

CD56+ cells in canine blood leukocytes were characterized by flow-cytometric analysis of peripheral blood of 30 healthy adult beagle-dogs (15 males and 15 non-pregnant females). In 19 of the 30 dogs, anti human CD56 antibody, Leu-19, reacted with 8.8-21.7% of peripheral blood lymphocytes. All CD56+ cells simultaneously expressed CD3 molecules on their surface. Further phenotypic analysis revealed that 50.6+/-13.1% of the CD56+ cells showed CD4-CD8+ phenotype and 43.7+/-10.1% showed CD4+CD8- phenotype. Expression intensity of CD56 on the CD4-CD8+CD56+ cells was significantly higher than that on CD4+CD8-CD56+ cells (P<0.001). These findings indicate that CD56, which is a neural cell adhesion molecule, is uniquely expressed on subsets of T lymphocytes in canine peripheral blood.     

Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement

BACKGROUND: Uniquely rearranged immunoglobulin and T-cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. OBJECTIVE: The purpose of this study was to determine whether the polymerase chain reaction (PCR) detects neoplastic (clonal) lymphocytes more frequently than do microscopic methods. METHODS: We identified neoplastic lymphocytes in peripheral blood by both routine and standardized microscopic examination of blood smears and by PCR amplification of blood-derived DNA and compared the 3 methods for frequency of detection of leukemic involvement. For standardized microscopic examination (200 leukocytes counted on Wright-Giemsa-stained blood smears), samples were categorized as negative (1% prolymphocytes, no lymphoblasts), or positive (>/=1 lymphoblast). A PCR-amplified sample was positive if 1 or 2 discrete bands were seen on the gel, or negative if no bands, a smear, or a faint ladder was seen. RESULTS: Using PCR, neoplastic lymphocytes were detected in peripheral blood 2.5 times more frequently than with routine or standardized microscopic evaluation. Eighty-three percent of samples negative by microscopy were positive by PCR. CONCLUSION: PCR is more sensitive than microscopy for the detection of clonal lymphocytes in peripheral blood. The results of this study also suggest that neoplastic lymphocytes circulate in peripheral blood at a higher frequency than previously reported. PCR may be useful for detecting or phenotyping lymphoma, monitoring response to therapy, identifying recurrence, and screening breeds at risk.     

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.     

Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue

Four wethers and 14 steers (environmentally heat stressed, 32 degrees C, 60% relative humidity) were evaluated for changes in blood flow induced by endophyte-infected tall fescue. Concentration of the ergopeptide ergovaline was used as an indicator of diet toxicity due to the endophytic fungus Acremonium coenophialum. Blood flow to specific tissues was measured using radiolabeled microspheres. Wethers received one of two dietary treatments for 30 d before determination of tissue blood flow: 1) a low-endophyte diet (less than .05 ppm ergovaline) or 2) a high-endophyte (1.18 ppm ergovaline) diet. Blood flows to the adrenal glands and skin covering the inner hind leg were less (P less than .10) in wethers consuming the high-endophyte diet than in those consuming the low-endophyte diet. Tissue blood flows in steers were determined on two occasions: 1) after steers had received a low- (less than .01 ppm ergovaline) or high-endophyte (.52 ppm ergovaline) fescue diet for 14 d and 2) 8 d after steers had been switched to a common, fescue-free diet. Blood flows to skin covering the ribs, cerebellum of the brain, duodenum, and colon were less (P less than .10) in steers consuming the high-endophyte diet. However, 8 d after consuming fescue-free diets, steers that had previously consumed the high-endophyte diet had greater (P = .08) blood flow to the coronary bands of the front hooves than steers that had consumed the low-endophyte diet. Blood flows to all other tissues were similar between treatments. We inferred from these experiments that the toxin(s) associated with endophyte-infected tall fescue caused decreased blood flow to peripheral and core body tissues and that this effect was abated within 8 d of removing the toxin(s).