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1.
The purpose of these investigations was to develop a modified whole blood technique for measuring quantitatively the responsiveness of pig peripheral blood lymphocytes to phytohaemagglutinin (PHA) in vitro. Washed blood cells from a fixed volume of blood were suspended in culture medium supplemented with foetal calf serum and stimulated with a pure mitogenic PHA preparation. The stimulation was measured by the incorporation of 3H-thymidine.Data are presented to show the effect of different variables on the culture system. The transformation response was measured at different levels of PHA and the reproducibility of the different responses from repeated investigations was used to evaluate the usefulness of the test.The best reproducibilities of the stimulation response were found at the high PHA concentrations.Also the calculated PHA concentration giving maximum stimulation response had a relatively high reproducibility and indicates that this value is a convenient and reliable alternative measure of the functional capacity of the lymphocytes.  相似文献   

2.
Bovine lymphocyte populations were characterized by surface markers, rosette-forming ability and behaviour towards mitogens. After pre-treatment with neuraminidase 16% of the bovine blood lymphocytes and 14% of the bovine spleen cells formed spontaneous (E) rosettes with sheep erythrocytes. About 20% EAC rosette-forming cells were detected among both cell populations. Protein A receptors were detectable among 8% of the blood lymphocytes and 26% of the spleen cells. Bovine lymphocytes responded to pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (Con A). An enrichment of bovine B and T cells was obtained by E-rosette sedimentation (81–84% B cells) and by filtration through nylon fiber columns (51–65% T cells). The T cells obtained after nylon filtration still responded to the mitogens PHA, Con A and PWM. Enriched B-cell populations responded to bacterial lipopolysaccharide (LPS). After monocyte depletion the mitogenic response of blood lymphocytes was not influenced.  相似文献   

3.
The effect of viable Mycoplasma bovis on the in vitro bovine peripheral blood lymphocyte response to phytohemagglutinin (PHA) was studied. Results showed that M. bovis did not act as a mitogen for bovine lymphocytes. Viable M. bovis produced a dose and time dependent suppression of the PHA stimulated lymphocyte response. Suppression was not a result of differences in the viability of infected or control lymphocyte cultures. The suppressive effect of M. bovis was found to be independent of the concentration of PHA used in the test and the lymphocyte response could not be restored by supplementation of the culture medium with arginine. Delay for 48 h after PHA stimulation before adding M. bovis to the lymphocyte cultures diminished, but did not prevent, the suppression of the lymphocyte response. These results show that suppression of the lymphocyte response does not require the presence of M. bovis during the period of PHA stimulation, and that M. bovis was capable of interrupting [3H]-thymidine incorporation in lymphocytes which were actively synthesizing DNA.  相似文献   

4.
In vivo inoculation of three-month-old calves with sodium diethyldithiocarbamate (DTC), killed Corynebacterium parvum or mycobacterium cell wall extract (MCWE) resulted in an enhancement of in vitro peripheral blood lymphocyte blastogenic responses to mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) in the first three days after treatment. In a separate experiment, blood lymphocytes isolated from a healthy nontreated calf were incubated in vitro in presence of each of the same immunostimulating agents and tested for their blastogenic responses to PHA and Con A. The results showed that all immunostimulants, excepting DTC, enhanced the in vitro blastogenic responses of lymphocytes to PHA and Con A. Finally, addition of MCWE to cultures of blood lymphocytes isolated from calves vaccinated intramuscularly with bovine rotavirus and adjuvant resulted in an enhancement of the in vitro lymphocyte transformation to rotavirus. Our study demonstrated that DTC, killed Corynebacterium parvum and mycobacterium cell wall extract were able to enhance bovine T cell proliferation in vitro.  相似文献   

5.
Culture medium from bovine peripheral blood mononuclear cells stimulated with the mitogens phytohaemagglutinin (PHA) or Concanavalin A (Con A) was found to maintain the proliferation of Con A blasts in vitro. The factor responsible for this activity was not absorbable with bovine erythrocytes or fresh peripheral blood lymphocytes but was removed by Con A blasts. Production of this factor was dependent on the dose of mitogen used and was greatest after 24 h culture compared to 48 h. Quantitative determinations of factor activity in supernatants were carried out by regression analysis of logit transformed data from assays measuring the maintenance of Con A blast proliferation by supernatants.  相似文献   

6.
Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentrations of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indices neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37 degrees C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubation in cell-culture medium between normal and atopic dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type 1 hypersensitivities such as atopy, can modulate lymphocyte function. At 10(-4) and 10(-8) M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H1 and H2 antagonists, we concluded that histamine enhanced mitogenesis via H1, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.  相似文献   

7.
The percentage of E rosette forming cells amounted to 26% of the blood lymphocytes and 34% of the spleen cells in German Landrace pigs. 10% of the live lymphocytes in the peripheral blood and 22% of the spleen cells were EAC rosette forming cells. The number of E rosettes could be increased by treatment of sheep erythrocytes with neuraminidase. The number of lymphoid cells reacting with protein A in the peripheral blood and in the spleen of pigs correlated well with the number of EAC rosette forming cells. The mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are potent stimulators of pig lymphoid cells. The mitogenic stimulation of pig lymphocytes could not be influenced significantly by the removal of phagocytic cells. By neuraminidase treatment the mitogen induced stimulation rate was decreased. For the mitogenic stimulation of porcine lymphoid cells in the presence of PHA, Con A and PWM T cells were required. Bacterial lipopolysaccharides (LPS) stimulated only B cells to a small degree.  相似文献   

8.
The influence of allogeneic IgG on in vitro reactivity of peripheral blood lymphocytes (PBL) of neonatal colostrum-deprived piglets as well as of suckling and weaned piglets was studied. PBL were preincubated with purified allogeneic IgG for 24 h before their ability to respond to PHA, Con A or PWM was tested. PBL of precolostral piglets pretreated with allogeneic IgG exhibited higher response to PHA (P less than 0.01) than untreated control cells. An increased response of PBL treated with IgG was also observed in suckling piglets as compared to their respective control cells (P less than 0.01). Responsiveness of PBL treated with IgG to PWM was suppressed. No differences in response to Con A regardless of the sources of lymphocytes was observed as compared to IgG untreated controls. The results suggest that pretreatment of lymphocytes of piglets with allogeneic IgG modulates their reactivity to mitogens, suppressing the response to PWM and stimulating the response to PHA, respectively.  相似文献   

9.
A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blastogenic responses of lymphocytes from twenty mares were observed during various stages of the perinatal period. Response decreased gradually before parturition was lowest at the time of parturition (PHA: 1.923 +/- 0.174, Con A: 1.698 +/- 0.206 and PWM: 1.706 +/- 0.177), and then increased gradually after parturition towards non-pregnant levels.  相似文献   

10.
A comparison of the mitogenic effects of a phorbol ester on lymphocytes from bovine spleen, lymph node and peripheral blood.Bovine lymphocytes from three tissues, lymph node, spleen and peripheral blood were compared for their mitogenic responses to 12-O-tetraecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter. TPA alone was found to be either not mitogenic or caused only a weak response when compared with the mitogenic lectin phytohemagglutinin (PHA). Of the three lymphocyte preparations, blood cells showed the greatest proliferative response to TPA. However, all three, lymph node, blood and spleen cells, showed a co-mitogenic response to TPA. That is, TPA synergistically enhanced DNA synthesis in cells stimulated with a suboptimal concentration of PHA.  相似文献   

11.
Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled (32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweek mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.  相似文献   

12.
The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6 degrees C, 8 x 10(5) cells in 200 microliters medium (= 4 x 10(6) cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 micrograms/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 microCi [3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 micrograms/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 micrograms/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.  相似文献   

13.
Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.  相似文献   

14.
Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavlin A (Con A), pokeweed mitogen (PWM), and pl ytohemagglutinin (PHA) at optimal concentrations (Con A, 15 μg/ml, PWM and PHA, 5 μg/ml) and decreased concentrations (Con A, 5 μg/ml, PWM and PHA, 1 μg/ml). Mitogenesis induced by lipopolysaccharide was considerably smaller and not used routinely. With 2 × 105 lymphocytes/well, the background count of unstimulated lymphocytes in autologous serum in healthy dogs was usually between 100 and 400 counts/min (CPM), in clinically healthy cattle and horses from 200 to over 2000 CPM. Higher CPM were rarely detected without clinical disease. Increased background counts were often associated with viral infections, leukemias and lymphoreticular hyperplasias, decreased background counts were associated with various diseases. The stimulation indexes (SI) of healthy animals in autologous serum with Con A, (5 μg/ml) or PWM or PHA (1 μg/ml) were in the range from 100 to 1000 in the dogs, in the tens for Con A and in hundreds for PWM and PHA in horses and cattle. Increased SI were present during the incubation period of various diseases. Decreased SI were associated with numerous infectious and lymphoreticular diseases and were caused by any of the following: (1) the presence of serum immunosuppressive factor(s) in the patient serum, (2) the decreased response of lymphocytes to mitogens, or (3) increased mitogenicity of lymphocytes due to unidentified serum factors in absence of phytomitogens.  相似文献   

15.
We have found that various sorts of virus particles, including avian leukosis and sarcoma viruses, Sendai virus, Friend leukemia virus and murine mammary tumor virus, are able, upon coincubation with chicken peripheral lymphocytes and either Concanavalin A (Con A) or phytohemagglutinin (PHA), to inhibit the mitogenic responses that normally follow. Such inhibition is not dependent on the use of infectious virus, and can be documented by using particles whose infectivity has been abolished by irradiation with ultraviolet light. Lymphoid cells that had been preincubated with viruses at concentrations approximating 10 particles per cell and for only 2 hours were inhibited by as much as 83% in ability to respond to the lectins employed. These results appear to be mediated, at least in part, by a virus-induced factor with the capacity to abrogate the responsiveness of freshly obtained lymphocytes to mitogenic stimulus. This factor is devoid of interferon activity, as tested in a vesicular-stomatitis-virus plaque reduction assay.  相似文献   

16.
Peripheral lymphocyte function in dogs with Brucella canis infection was evaluated using in vitro lymphocyte stimulation with the mitogens PHA, Con A and PWM, and killed Brucella canis organisms. Bitches with naturally occurring Brucella canis infection were compared to negative controls. There was no difference in the response to Con A and PWM between these two groups. Lymphocytes from infected dogs were less responsive (p less than .05) to PHA than were lymphocytes from controls. There was a significant (p less than .005) difference in response to Brucella canis antigen between the two groups. Lymphocytes from infected dogs were stimulated by Brucella canis antigen, whereas those from controls did not respond.  相似文献   

17.
Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.  相似文献   

18.
Characterization of monoclonal antibodies directed against swine leukocytes   总被引：3，自引：0，他引：3  
Hybridomas were produced from fusions of the SP2/0 mouse myeloma with splenic cells from: 1) an outbred Sprague Dawley rat immunized with swine peripheral blood mononuclear (PBM) cells; 2) a (CBA/NDub X BALB/c Dub) F1 mouse immunized with concanavalin A (Con A) activated swine PBM cells and 3) a (BALB/c Dub X C3H/He Dub) F1 mouse immunized with swine thymocytes. The resulting supernatants were screened by a microcytotoxicity assay for activity against swine PBM cells. Four hybridomas (MSA1, MSA2, MSA3 and MSA4) were selected, cloned and characterized by their cell reactivity and effect on mitogenic assays. MSA1 and MSA2 belong to the rat IgG2b subclass. MSA3 and MSA4 are of the mouse IgG2a subclass. These monoclonal antibodies reacted in the following manner: MSA1 with monocytes, granulocytes, red blood cells and bone marrow cells; MSA2 with subset of T cells; MSA3 with B cells and subsets of T cells and monocytes (class II molecule) and MSA4, a pan-T cell reagent (E-rosette receptor). The involvement of the various cell types reactive to the different monoclonal antibodies in the mitogenic response of swine PBM cells to Con A, phytohemagglutinin (PHA) or pokeweed mitogen (PWM) was investigated by cellular depletion with monoclonal antibody plus complement. Cellular depletion of PBM cells with the following monoclonal antibodies plus complement treatment resulted in: MSA1, almost total reduction in the mitogenic response to low doses of Con A or PWM; MSA2, partial reduction in the proliferative responses to any concentration of Con A, PHA or PWM; MSA3, partial reduction in proliferative responses to low concentrations of Con A or PWM and 4) MSA4, total elimination of any proliferative response to Con A, PHA or PWM.  相似文献   

19.
Characterization of lymphocyte subpopulations in the bovine mammary gland was accomplished using cells obtained from dry secretions. Correlation of cell surface properties with functional capacity was attempted by assaying the ability to form erythrocyte-antibody (EA) rosettes, erythrocyte-antibody-complement (EAC) rosettes, and sheep erythrocyte (E) rosettes and the ability to respond to phytohemagglutinin (PHA), Concanavalin A (Con A), and pokeweed mitogen (PWM) in the lymphocyte stimulation test. Results were compared with those obtained for peripheral blood lymphocytes (PBL) from the same animals. Mammary gland lymphocytes (MGL) formed significantly fewer (p < .01) EA and EAC rosettes, but significantly greater (p < .01) E rosettes compared to PBL. MGL were significantly less responsive (p < .05) to mitogens than were PBL. MGL contained a large proportion of T lymphocytes, which do not respond to T lymphocyte mitogens in culture.  相似文献   

20.
Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H3]-thymidine ([H3]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 105 viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H3]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.  相似文献   

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