Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.     

Relationship between mitogen receptors in peripheral blood lymphocytes and blastogenic responses to mitogen

The relationship between the optimum concentration of mitogen which induces lymphocyte blastogenic response and the receptor occupancy by mitogen was investigated. The receptor occupancies which induced maximal blastogenic activity in equine, bovine and canine peripheral blood lymphocytes (PBL) were 31.1 per cent, 26.5 per cent and 38.4 per cent with phytohaemagglutinin-P, and 48.2 per cent, 17.9 per cent and 24.5 per cent with concanavalin A, respectively. The data clearly show that each animal species had its own optimum concentration of mitogen for stimulation of PBL. Optimum concentration for blastogenesis and number of binding sites of each mitogen had a good correlation with each other for all three species.     

A micromethod for evaluating turkey lymphocyte response to phytomitogens.

A microculture system used in conjunction with a semiautomatic sample harvester is described to determine the in vitro properties of turkey peripheral blood lymphocytes. By this new procedure, multiple tests were done rapidly, relatively few cells were used, and results were highly reproducible. Analysis indicated that the conditions for optimal concanavalin A (con A) stimulation, as measured by incorporation of 3H-thymidine (3H-TdR) included (a) 2 times 10(6) cells per culture in RPMI 1640 medium in the absence of any serum; (b) 0.4 mug of con A per culture, incubated in flat-bottom microtitration wells for 72 hours at 37 C; and (c) 1 muCi of 3H-TdR per culture added 12 to 24 hours before termination. Conditions for optimal stimulation with pokeweed mitogen were similar to those used for con A. The exceptions were that 1 times 10(6) cells per culture were incubated in round-bottom microtitration wells. The pokeweed mitogen gave the highest degree of stimulation when used at a concentration of 80 mug per culture.     

Bovine leukosis virus in sheep,lymphocyte modification and surface-immunoglobulin-bearing cell numbers

Lymphocytes from sheep experimentally infected with bovine leukosis virus (BLV) and from non-infected normal sheep were examined for the presence of surface Ig by an immunofluorescence test. Surface Ig-bearing lymphocytes in blood from BLV-infected sheep increased when lymphocyte counts of blood were elevated in comparison with normal animals. The mitogen stimulation of cultured lymphocytes from BLV-infected sheep and from non-infected normal sheep was determined by measuring ³H-thymidine incorporation. Peripheral blood lymphocytes (PBL) from BLV-infected leukemic sheep with elevated PBL counts responded poorly to phytohemagglutinin M and concanavalin A but responded well to lipopolysaccharide compared with lymphocytes from uninfected animals. In BLV-infected preleukemic sheep with low PBL counts, stimulation indices of mitogen responses of lymphocytes with phytohemagglutinin M, concanavalin A, and pokeweed mitogen were low compared with those of lymphocytes from uninfected animals. The results indicated that B cells were affected by BLV infection in sheep as suggested by the increased number of surface Ig-bearing lymphocytes and that significant alteration of mitogen stimulation of lymphocytes occured in sheep with BLV infection.     

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.     

In vitro blastogenic responses of peripheral lymphocytes from marmosets to phytohemagglutinin and to autologous lymphocytes transformed by Epstein-Barr virus

White-lipped marmosets were evaluated for their cell mediated immune (CMI) response to EBV to determine the feasibility of CMI studies in marmoset models for EBV oncogenesis. The mitogen, cell concentrations, the length of incubation period and serum requirements were defined for in vitro lymphocyte stimulation tests. The level of response of each animal was dependent on the concentration of phytohemagglutinin-P (PHA-P) and was independent of cell densities employed. The rate of tritiated-thymidine incorporation by mononuclear cells due to PHA-P increased exponentially between 2–4 days. This test was reproducible for a given batch of PHA-P when the cells were cultured in the presence of 10% heat inactivated fetal bovine serum. The five white-lipped marmosets were seronegative for EBV antigens and did not show lymphocyte stimulation with EBV particles and EBV soluble antigen, but two of these animals exhibited significant stimulation with autologous lymphocytes transformed in vitro by B95-8 virus. Despite the limited amount of blood (3–4 ml) that could be obtained from each animal in a single bleeding, it was possible to perform multiple lymphocyte stimulation assays with the protocol used.     

The influence of regular physical activity on the cell-mediated immunity in pigs

The influence of moderate regular physical activity on the cell-mediated immunity was studied in growing pigs. Ten animals were subjected to physical training on a large animal treadmill, and 10 were kept in their pens throughout a 12-week experimental period. Regardless of whether the pigs underwent training or not, a whole blood lymphocyte stimulation test performed at 3 stages of the experiment revealed an equal ability of the cells to respond to stimulation induced by pokeweed mitogen and phytohaemaglutinin. The influence of serum from the pigs of the trained and untrained groups was studied in a stimulation test with purified mononuclear cells obtained from 2 healthy control pigs. The results indicated that no additional serum factors released by the physical training altered the blastogenic response of these lymphocytes. It is concluded that moderate exercise should not be regarded as a stressor which alters the cellular immunity in pigs.     

Short-term effect of cyclophosphamide and azathioprine on selected aspects of the canine blastogenic response

The short-term, in vitro responses of canine peripheral blood lymphocytes to mitogenic stimulation and serum immunoglobulin concentrations were evaluated following treatment with currently recommended doses of cyclophosphamide and azathioprine. Cyclophosphamide had no significant effect on either the serum immunoglobulin concentrations or the blastogenic response of lymphocytes to mitogenic stimulation. Serum immunoglobulin concentrations remained unchanged following azathioprine treatment. The blastogenic response was significantly suppressed following one week of azathioprine therapy and returned to baseline values one week following cessation of treatment. The response to phytohemagglutinin was most suppressed, followed, in order, by the response to concanavalin A, and to pokeweed mitogen. These results suggest that the short-term use of azathioprine, but not cyclophosphamide, in clinically used dosages, does suppress selective aspects of the canine immune system, and the T cells appear to be more susceptible than B cells to the immunosuppressive effect of this drug.     

Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal disease virus

Chickens were inoculated wih the pathogenic Edgar strain of infectious bursal disease virus at 1 week, 2 weeks, or 1 day of age. In the 3 experiments, phytohemagglutinin stimulation of peripheral blood lymphocytes was significantly decreased on day 3 or 4 after inoculation. Subsequently, on days 7 through 21, stimulations were similar between lymphocytes from inoculated birds and those from control birds. Pokeweed mitogen stimulation was affected minimally in virus-inoculated chickens. In each experiment, on day 7, the spontaneous [3H]thymidine uptake was greater in nonstimulated lymphocyte cultures from inoculated chickens than in such cultures from control chickens. In an additional experiment, chickens 1 week of age were exposed to a pathogenic vaccinal virus given in their water. The vaccinal virus exposure resulted in significant decrease of phytohemagglutinin stimulation of lymphocytes on days 3 and 7 of the experiment. A significant decrease in pokeweed mitogen stimulation was observed on day 10 after inoculation.     

Duck lymphocytes--III. Transformation responses to some common mitogens

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.     

酶解鸡蛋清小肽混合物对小鼠免疫功能的影响

本研究考察了鸡蛋清经Flourezyme(FL)、Alcalase(AL)、胃蛋白酶(PE)、胰蛋白酶(TR)或木瓜蛋白酶(PA)5种酶水解后制备的5种酶解小肽混合物(MSEE)对小鼠体外免疫功能的影响。采用体外培养,细胞吞噬实验和细胞增殖实验,观察不同种类及不同剂量的MSEE对小鼠腹腔巨噬细胞吞噬能力和脾细胞增殖能力的影响。结果发现,不同种类的MSEE均能显著(P<0.05)或极显著(P<0.01)提高巨噬细胞的吞噬能力,促进淋巴细胞的增殖,且表现出一定的剂量效应关系;1mg/mL不同MSEE的促吞噬能力大小顺序为:FL>PA>AL>PE>TR>对照,促脾淋巴细胞增殖能力大小顺序为:FL>PE=AL>TR>PA>对照(无丝裂原),FL>AL>PA>TR>PE>对照(LPS刺激),FL=AL>PE>PA>对照>TR(ConA刺激)。这些结果表明,5种MSEE均具有免疫增强活性,其中FL的活性最强。     

A study of porcine lymphocyte populations. II. Characterization of porcine lymphocyte populations

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.     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Thyroid and immunologic status of dogs with perianal fistula

Lymphocyte-proliferation responses, absolute lymphocyte counts, and thyrotropin-stimulation responses were determined in 33 dogs with perianal fistula; serum immunoglobulin values also were determined in 15 of the 33 dogs. Lymphocytes were stimulated with concanavalin A, pokeweed mitogen, and phytohemagglutinin and were cultured with medium containing normal pooled canine serum or fresh patient's autologous serum. Initially, lymphocytes from 9 dogs (27.3%) had depressed stimulation responses to greater than or equal to 1 phytomitogen, and 4 of the 9 dogs had absolute lymphopenia. One month after recovery in these 9 dogs, lymphocytes from 4 dogs (66.7%) had normal proliferation responses. Of immunoglobulin determinations in 15 dogs, serum IgA values were 32 to 185 mg/dl (mean, 69 +/- 10 mg/dl) and were low in 2 dogs (13%), and serum IgM values were 48 to 610 mg/dl (mean, 263 to 46 mg/dl) and were high in 8 dogs (53%). Serum IgG values were 1,050 to 3,220 mg/dl (mean, 2,339 +/- 165 mg/dl) and were high in 10 dogs (71%). After thyrotropin stimulation, 1 dog was considered hypothyroid. Neither pathogenesis nor prognosis of canine perianal fistula was clarified via immunoglobulin concentrations or absolute lymphocyte counts. Based on lymphocyte-proliferation assays, suppression of cell-mediated immunity was probably a result of perianal fistula, rather than a cause of the fistula.     

Phytomitogen- and antigen-induced blast transformation of feline lymphocytes.

A semiautomated microassay of blast transformation of feline lymphocytes was developed and used in a study of the response of normal cats to phytomitogen- and antigen-induced stimulation. Peripheral blood lymphocytes were isolated from defibrinated blood by ficolldiatrizoate gradient centrifugation and incubated at a concentration of 1 X 10(5) cells/culture. Blast transformation was measured by the incorporation of tritiated thymidine, to which the cells were exposed during the last 18 hours of incubation. Phytomitogens induced maximal transformation after 3 days' incubation. Greatest stimulation produced by 10 mug of concanavalin A (38- to 183-fold) followed by 4 mug of pokeweek mitogen (nine- to 51-fold), and finally phytohemagglutinin-P (three- to sixfold). Three of 4 cats sensitized to keyhole limpet hemocyanin responded when exposed to 50 mug of keyhole limpet hemocyanin in vitro, maximal transformation occurring after 4 days' incubation. Lymphocyte blast tranformation provides a convenient technique to assess lymphocyte function serially during experimentally induced immunologic alterations in the cat.     

Effect of levamisole on immune function and reproductive performance in first-litter gilts

First-litter commercial cross-bred gilts were treated with levamisole (1.5, 2.5, or 3.5 mg/kg of body weight) weekly during the last 4 weeks of gestation, because similar treatment of dairy heifers had improved postpartum maternal health and neonatal survival. In the gilts, differences in reproductive performance were not found on the basis of pig survival at birth, pig survival at weaning, birth weight, or weaning weight. Also, differences between treated and control gilts were not found in response of circulating lymphocytes to mitogen stimulation (phytohemagglutinin A, concanavalin A, and pokeweed mitogen). In all gilts, the lymphocyte response to mitogen stimulation was decreased during the first week after farrowing.     

Stimulation by phytohaemagglutinin of peripheral blood lymphocytes from horse,pig, sheep and man

Optimal conditions for stimulation by phytohaemagglutinin (PHA) were established for equine, porcine, ovine and human lymphocytes in MEMS medium. Optimal thymidine concentration was determined for assay of cell transformation. With all species tested horse serum gave highest thymidine incorporation. Homologous serum was not more appropriate for lymphocytes of man, pig and sheep. Optimal stimulation was achieved at 20, 0.5–5, 5, and 10–40 μg PHA per 10⁶ cells for human, equine, porcine and ovine lymphocytes, respectively.     

Selective IgM deficiency and abnormal B-cell response in a foal.

Selective IgM deficiency was diagnosed in a 3-month-old Standardbred colt that was referred for chronic respiratory tract disease. Immunoglobulin quantification revealed normal IgG and IgA concentrations, but undetectable IgM concentration. Stimulation of blood lymphocytes with the T-cell mitogens concanavalin A and phytohemagglutinin yielded results within the normal range. However, stimulation with the B-cell mitogen lipopolysaccharide produced no response. A B-cell defect similar to that associated with several immunodeficiency disorders in people was suggested as the cause of the IgM deficiency in this colt.     

Lymphocyte blastogenesis in bluetongue virus or Mycobacterium bovis-inoculated bovine fetuses

Bovine fetuses were inoculated with Mycobacterium bovis, bluetongue virus or placebo at approximately 125 days of gestation, and blastogenic responses of peripheral blood lymphocytes and lymph node cells were determined at various time intervals after inoculation. Lymphocytes from all fetuses were stimulated by phytohemagglutinin, concanavalin A, and pokeweed mitogen, and peripheral blood lymphocytes gave consistently greater stimulation indices than did prescapular lymph node cells. Bluetongue virus infection did not consistently suppress mitogen induced lymphocyte blastogenesis. Lymphocytes taken from fetuses at 20 or 50 days after Mycobacterium bovis inoculation were not stimulated by purified protein derivative (PPD), whereas lymphocytes taken from adult cattle at similar intervals after Mycobacterium bovis inoculation were stimulated by PPD. Although lymphocytes from bovine fetuses may be stimulated by mitogens, antigen specific blastogenesis to a known inducer of cellular immunity was not detected by 175 days of gestation.     

Changes in lymphocyte blastogenic response of mares during the perinatal period

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.