Technical aspects of low 3H-thymidine incorporation by mitogen stimulated canine peripheral blood lymphocytes in vitro

The value of [3H]-thymidine incorporation as a measurement for mitogen induced proliferation of dog peripheral blood lymphocytes (PBL) has been examined. The cells were cultured in RPMI 1640, enriched with 10% autologous plasma for 48 hours at 37 degrees C, 5% CO2 and 95% relative humidity. Under these conditions a great variability in [3H]-thymidine incorporation was observed. By analysis of CPM and number of activated cells (G1), it was found that comparable number of G1 cells were generated in human and dog PBL. Also, the membrane transport of thymidine was very similar for lymphocytes of the two species. Nevertheless, a low [3H]-thymidine incorporation by dog PBL was frequently seen, and this phenomenon could be related to a release of soluble substance(s) within the cultures. When the cultured cells were washed and resuspended in fresh medium immediately before pulsing, the expected CPM per G1 cell could be obtained. Since it has been described in the literature that macrophages can produce cold thymidine in macrophage enriched lymphocyte cultures, the in vitro response of non-adherent dog PBL was analyzed. Mitogen stimulation of such non-adherent cells resulted in CPM per G1 cells very similar to those obtained with washed cells. Based on these data, it is suggested that the production of cold thymidine might be one of the technical problems related to cultures of lectin stimulated dog PBL in vitro and it should be taken into consideration, if [3H]-thymidine incorporation is used as the only measure of lymphocyte proliferation.     

Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (type I trophoblast interferon) and bovine interferon-alpha I1.

Bovine trophoblast protein-1 (bTP-1) is a Type I interferon secreted by the bovine trophoblast from about Day 15 of pregnancy. It is not known whether bTP-1 has functional properties in common with other interferons. The aim of the present study was to determine whether bTP-1 inhibits proliferation of lymphocytes induced by mitogens, mixed lymphocyte cultures (MLC) and interleukin-2 (IL-2) and, if so, whether this activity is similar to that of a related interferon, bovine interferon-alpha I1 (bIFN-alpha I1). Stimulation of lymphocyte proliferation caused by phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) was inhibited by bTP-1 and bIFN-alpha I1 without any reduction in cell viability. Maximum or near-maximum inhibition (less than 50%) was achieved at concentrations of 0.5-5.0 nM of bTP-1 and bIFN-alpha I1. Cells stimulated with PWM were less inhibited than cells stimulated with PHA and Con A. Both bTP-1 and bIFN-alpha I1 inhibited MLC to a greater degree than lectin-stimulated cells (maximum inhibition was 78% or greater). Also, bTP-1 and bIFN-alpha I1 slightly inhibited incorporation of [3H]thymidine ([3H]TdR) induced by the combination of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), and calcium ionophore A23187. Finally, bTP-1 and bIFN-alpha I1 had bimodal effects on incorporation of [3H]TdR by IL-2-induced lymphocytes. Incorporation of [3H]TdR was increased at 0.005 nM and 0.05 nM concentrations while higher concentrations caused a slight decrease in [3H]TdR incorporation. Results confirm that bTP-1 inhibits lymphocyte proliferation in a manner similar to that caused by the leukocyte-derived interferon, bIFN-alpha I1. Incomplete inhibition of mitogen-induced proliferation and differences in degree of inhibition between various stimulators suggest that bTP-1 and bIFN-alpha I1 preferentially inhibit certain lymphocyte subpopulations. Local inhibition of lymphocyte proliferation caused by bTP-1 may help protect the allogeneic conceptus from immune responses to fetal antigens or regulate the release of cytokines from endometrial lymphocytes.     

Comparison of intradermal injections of bromodeoxyuridine and tritiated thymidine in the in vivo measurement of epidermal cell turnover time in goats.

Study of keratinisation defects such as seborrhoea may require investigation of epidermal cell kinetics in clinical cases. For this to be practically useful a safe, reliable and quick method is essential. This study compared epidermal cell kinetics in the goat following the use of intradermal injection of tritiated thymidine ([3H]TdR) and bromodeoxyuridine (BURD) to label S phase nuclei. Turnover time for goat epithelial cells was not significantly different for the two agents; 26.3 +/- 6.0 days for thymidine and 21.8 +/- 5.2 days for BURD. It was concluded that intradermal injection of BURD will be a suitable technique for investigating epidermal cell kinetics in vivo and could have applications in a clinical situation because it is quicker and safer than [3H]TdR.     

Effects of ionizing radiation on in vitro proliferative responses of porcine peripheral blood lymphocytes

The radiosensitivity of $\text{in vitro}$ proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine $\text{in vitro}$, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary $\text{in vitro}$ proliferative response.     

African hamster lymphocytes as a model for immunologic studies.

The mitogenic response of splenic lymphocytes from Mystromys albicaudatus was studied to evaluate the potential of this animal as a model for immunologic research. In response to phytochemagglutinin and concanavalin A, splenic cells from Mystromys, unlike those from mouse strains, incorporate [3H] thymidine optimally in microculture at 10(5) to 2 X 10(5) cells per microculture. Maximum magnitude of incorporation in response to the same doses of mitogen used in mouse splenic lymphocyte microculture is greater than 10(5) cpm. Moreover, this high incorporation at low cell concentration has been observed in cultures from animals ranging from six to 24 months of age. Splenic cells from Mystromys give little or no incorporation with either LPS or PPD in doses mitogenic to mouse lymphocytes. These features of mitogenic response in Mystromys lymphocyte cultures suggest several useful applications to studies of mechanisms of mitogenesis.     

Immunomodulatory properties of a strain of Mycobacterium chelonae. I. Mouse lymphocyte responses in vitro

Immunomodulatory properties of a strain of live Mycobacterium chelonae (Mch) was investigated in an in vitro lymphocyte transformation system. Murine splenocyte activation by this bacterium was characterized by polyclonal lymphoproliferative responses in a dose dependent fashion. Optimal doses ranging from 20 to 80 micrograms of Mch (wet weight) per ml of cell suspension induced a very significant mitogenic effect. Higher doses (100 micrograms) of Mch manifested a decreased rate of tritiated thymidine ([3H] TdR) uptake whereas responsiveness of splenic lymphocytes to lower doses (0.156 microgram) was not modified. Contrary to the splenocyte responses activation of murine thymocytes by this mycobacterium is characterised by a decreased proliferation as compared to the background count of unstimulated cells. Simultaneous addition of Mch with optimal doses of Concanavalin A (Con A) and Phytohemaglutinin (PHA) potentiated polyclonal mitogenic responses of murine splenocytes to these two lectins. However, proliferation of these lymphocytes to Lypopolysaccharide (LPS) induction was not modified. BALB/C and DBA/2 spenocytes were found to be more responsive to stimulation by this Mycobacterium as compared to those of C3H/Ou and to a lesser degree to those of C57BL/6 mice.     

Culture conditions for blastogenic responses of bovine mammary mononuclear cells

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.     

Canine lymphocyte cultures in vitro: Evaluation of peripheral blood lymphocyte response to mitogens

Lymphocytes from dog peripheral blood have been stimulated in vitro with 3 different mitogens (Con A, PHA and PWM). Culture medium was RPMI 1640 enriched with either autologous plasma, fetal calf serum or a newly described defined serum substitute. In such cultures the number of surviving and activated cells was measured by cytofluorometry and the proliferation was assessed by thymidine incorporation. In unstimulated cultures, up to 70% of all cells had disappeared (died) during the first 42 hours of incubation, whereas the number of viable cells was reduced to 50–60% in mitogen stimulated cultures. Of the surviving lymphocytes, between 25–40% of the cells appeared to have an elevated RNA-content (activated or G₁ cells). By comparison between thymidine incorporation and number of mitogen induced G₁ cells, a very high correlation was found (r=0.92). However, the slope of the regression line was much lower than expected. The low thymidine incorporation per activated cell was primarily related to the high cell death and a resulting dilution of tritiated thymidine. Indeed, preliminary results suggested that the same thymidine incorporation per G_1b cells could be obtained if peripheral blood lymphocytes were washed immediately before pulsing as could be obtained with lymphnode cells without washing.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

Insulin and insulin-like growth factor-I stimulate DNA synthesis in bovine mammary tissue in vitro

Effects of insulin and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation, in vitro, by mammary tissue slices obtained from prepartum and lactating cows were investigated. Both insulin and IGF-I induced up to a 10-fold increase in [3H]thymidine incorporation in the mammary slices cultured in serum-free media. The effect of insulin-stimulated [3H]thymidine incorporation occurred at a threshold of greater than 1.75 pmol/ml and appeared to reach maximum at greater than 8.8 nmol/ml. The response to IGF-I occurred at greater than 6.5 pmol/ml and reached the equivalent of maximal insulin-stimulated incorporation at 39 pmol/ml. No synergistic or additive effects were observed between these two factors. The in vitro response took 3 to 4 d to reach maximum and was inhibited by cytarabine. Mammary tissue obtained from lactating cows incorporated more [3H]thymidine per microgram DNA in response to insulin (175 pmol/ml) than mammary tissue from pregnant cows. Culture of mammary tissue slices with growth hormone, cortisol, prolactin, or triiodothyronine showed no stimulation of [3H]thymidine incorporation over control. Autoradiography of the cultured lactating tissue showed incorporation of [3H]thymidine by 51, 24 and 29% of the ductal epithelial, secretory alveolar epithelial and myoepithelial cells, respectively. All alveolar epithelial cells that incorporated [3H]thymidine contained secretory products. Among nonsecretory cells, 25 and 28% of the fibroblasts and white blood cells, respectively, were labeled. Insulin-like growth factor I, but not bovine somatotropin, stimulated [3H]thymidine uptake into DNA in lactating bovine mammary tissue. Thus, our data support the concept that bovine somatotropin acts through IGF-I to increase DNA synthesis in mammary cells.     

Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes

Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Flow Cytometric Analysis of Mitogen‐Induced Activation of Rainbow Trout (Oncorhynchus mykiss) Peripheral Blood Leucocytes

Proliferation of rainbow trout peripheral blood leucocytes in vitro is usually assessed by measuring incorporated tritiated thymidine. In this report we monitored the in vitro proliferative response to the mitogen Concanavalin A (Con A) by means of flow cytometry (FCM) and ³H‐thymidine incorporation. When analysed by FCM, blood leucocytes displayed two main cell populations with distinct forward and side scatter (FSC/SSC) characteristics: lymphocytes with low FSC/SSC values and non‐lymphoid leucocytes (NLL) with increased FSC/SSC values. The nature of these cell types were confirmed by microscopy. Interestingly, the FSC/SSC pattern of lymphocytes remained unchanged after in vitro stimulation with Con A, whereas cells from the NLL population showed a marked shift towards increased FSC values. In stimulated cultures, the increase of FSC values of the NLL population significantly correlated with contemporarily measured ³H‐thymidine incorporation (r=0.7, P < 0.001). The mitogenic response of blood leucocytes originating from different individual fish varied over wide ranges. It was found to be related to the numbers of NLL present in the leucocyte sample. The present results show that qualitative and quantitative FCM analysis of morphological parameters (FSC/SSC) of blood leucocytes makes it possible to discriminate between leucocyte populations of the rainbow trout and to monitor cell proliferation experiments.     

Development of immune responsiveness to Ascarissuum antigens in pigs vaccinated with ultraviolet-attenuated eggs

Pigs fed $\text{Ascaris}$$\text{suum}$ eggs attenuated by short wave ultraviolet-radiation developed resistance to challenge infection. Per os inoculation of pigs on three successive weeks with 10,000 eggs irradiated to total exposures of 150,100 and 75 μW-min/cm², respectively, resulted in an 88% reduction in the number of larvae recovered from the lungs 7 days after challenge with 10,000 infective eggs. Peripheral blood lymphocytes (PBL) from vaccinated pigs were specifically stimulated in vitro to incorporate tritiated thymidine by egg hatching fluid (Ea) and by excretory-secretory products obtained from cultures in defined-media of second-stage larvae (L2) developing to third-stage (L2–3) and from cultures of third-stage larvae developing to fourth-stage (L3–4). PBL were also specifically stimulated by living L2. Ea and L2 stimulated pig PBL significantly at 7 days after the first inoculation; responses to L2–3 and L3–4 developed 7 days after a second inoculation. The antigen-responsive cells in the PBL population were non-immunoglobulin-bearing lymphocytes. Antibodies to Ea and L2–3 were not detected in the serum of vaccinated pigs, and only 3 of 7 pigs had low concentrations of serum antibodies to L3–4.     

Induction of blastogenesis in bovine peripheral blood lymphocytes by oxidation with sodium periodate

Suitable treatment and culture conditions are defined for the induction of blast transformation in bovine peripheral blood lymphocytes by oxidation with sodium metaperiodate (NaIO4). Stimulation with NaIO4 required slight modification of techniques used routinely for activation of lymphocytes in vitro with lectins and antigens. Gradient-separated mononuclear leukocytes responded with maximal [3H]TdR incorporation after oxidation with 0.50 to 1.0 mM NaIO4 for 30 minutes at 25 C. Oxidized cells cultured at 1 to 2 X 10(6)/ml responded better than cells cultured at any other concentration, when compared with untreated cells. Blastogenesis in response to oxidation reached its maximum rate within 48 hours of treatment, after which it declined rapidly. Partial removal of glass wool-adherent cells reduced periodate-triggered blastogenesis by 95%, but did not significantly affect activation with phytohemagglutinin, concanavalin A, pokeweed mitogen, or purified protein derivative. Reintroduction of macrophages restored responses to their precolumn level. Oxidation with NaIO4 provided a simple, rapid means of inducing blastogenesis in bovine lymphocytes. Manipulation of the well-defined triggering conditions may help to explain the mechanisms involved in lymphocyte activation.     

Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.     

The inhibitory action of sodium butyrate on the growth of KB,MMT and RPMI cells

Three tumour cell lines (KB, MMT and RPMI) established from epithelial tissues were treated for 24 h with sodium butyrate (BU), the BU concentrations giving rise to 50% inhibition of [³H]thymidine incorporation were 2.0, 0.3, 0.2 mmol/L, respectively, for the KB, MMT and RPMI cell lines. Studies with [¹⁴C]BU have shown that, at similar degrees of inhibition of [³H]thymidine incorporation, the intracellular concentrations of BU are very close for all three cell types, despite the dissimilarity of the extracellular BU concentrations. These results imply that the BU sensitivity of the cells does not depend on the inhibition of thymidine incorporation, but on their BU uptake. The [³H]thymidine incorporation of MMT cells exposed to 5 mmol/L BU for 72 h returned to normal within the next 48 h. The same treatment accounted for about an 80–90% decrease in the cloning efficiency and tumourigenicity of MMT cells. These findings indicate that BU pretreatment inhibits DNA synthesis temporarily, while other parameters related to the cell growth, such as cloning efficiency and tumourigenicity, are durably influenced by BU pretreatment.     

Modulation of the cellular immune responses to T-cell-dependent and T cell-independent antigens in lambs with induced bovine viral diarrhea virus infection

Functional interaction between lymphoid cells and lymphotropic viruses is particularly evident for bovine viral diarrhea virus (BVDV) in cattle and its closely related virus, the border disease virus (BVDV) in sheep. The most important aspect of acute or chronic phases of BVDV or BDV infection was the host's increased susceptibility to secondary bacterial or viral infection. To study the ability of BVDV to alter the development of the cellular immune responses to concomitant inoculation with T cell-dependent and T cell-independent antigens, lambs were inoculated twice with rabbit RBC and Escherichia coli lipopolysacharide (LPS) and then were infected with a cytopathic strain of BVDV at postinoculation day 3. Leukopenia characterized by lymphopenia developed after BVDV infection. Increased [3H]thymidine incorporation was observed in resting or lectin-stimulated blood mononuclear cells in the first weeks after inoculation in BVDV-infected lambs, but was followed by decreased [3H]thymidine incorporation after the second inoculation for up to 8 weeks after initial inoculation. In contrast, transient decrease of blastogenic responses, associated with toxic effect of LPS, was detected in inoculated noninfected lambs, but was followed by stimulation of cellular immune responses. Inoculated noninfected lambs had good in vitro cellular immune response to rabbit RBC and LPS antigens, whereas lymphocytes from BVDV-infected lambs could not mount lasting cellular immune responses to antigens or BVDV. Results suggest that BVDV infection in lambs modulates the ability of lymphocytes to respond to lectins or antigenic stimuli according to the time after infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the 3H-thymidine incorporation assay.

To evaluate canine lymphocyte stimulation the radioactive thymidine incorporation assay is still the method of choice. In order to find a suitable non-radioactive alternative to the standard 3H-thymidine incorporation assay, proliferation of canine peripheral blood lymphocytes (PBL) was measured with three different colorimetric assays, using the two tetrazolium salts MTT and XTT and 5-bromo-deoxyuridine (BrdU). Isolated canine PBL were stimulated with two different mitogens, Concanavalin A (Con A) and Phytohemagglutinin (PHA), using different culture conditions. Applying statistical analysis we found that BrdU and MTT showed a high correlation to the 3H-thymidine incorporation assay, although the BrdU assay proved to be more sensitive than the MTT assay. No significant correlation between the XTT assay and the radioactive method was demonstrated. Consequently, the BrdU assay is the most suitable alternative to the radioactive method.