A comparative analysis of the mitogenic response of intestinal intraepithelial lymphocytes in various age groups of turkeys.

Mitogenic responsiveness of intestinal intraepithelial lymphocytes (i-IEL) to concanavalin A (Con A), phytohemagglutinin P (PHA-P), and lipopolysaccharide (LPS) from Salmonella typhimurium were evaluated in various age groups of turkeys by a colorimetric blastogenic microassay. Comparisons were made between mitogenic responses of turkey i-IEL and peripheral blood lymphocytes (PBL). The results from this study demonstrated that i-IEL and PBL of turkeys responded to T-cell mitogens, Con A and PHA-P, in every age group examined. The LPS induced a significant mitogenic response in PBL but not in i-IEL of turkeys. The mitogenic responses of turkey i-IEL and PBL to the three mitogens examined were similar to mitogenic responses observed in an earlier study performed by using chicken i-IEL and PBL. The results indicated a difference in mitogenic response between different age groups. An increase was found in mitogenic response of i-IEL to both T-cell mitogens from 3 days of age to 1 wk of age, whereas mitogenic response of PBL to all three mitogens declined significantly from 1 day of age to 3 days of age. The highest mitogenic response of i-IEL to T-cell mitogens was observed at 1 wk of age. The highest mitogenic response of PBL to both T-cell mitogens was observed at 1 day of age and the highest PBL response to LPS was observed at 16 wk of age. The mitogenic response induced by PHA-P provided less variability between age groups than the mitogenic response induced by Con A.     

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.     

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.     

Responses of enriched populations of feline T and B lymphocytes to mitogen stimulation.

Lymphocytes from healthy adult cats were separated into T and B cell-enriched subfractions by centrifuging rosetted cells on sodium metrizoate/Ficoll gradients. The responsiveness of unseparated lymphocytes (T + B), and T and B cell-enriched subfractions to stimulation with mitogens (phytohemagglutinin-P (PHA-P), concanavalin A (ConA), and pokeweed mitogen (PWM) was tested. Cultures of unseparated lymphocytes and those enriched in T cells showed similar responsiveness of PHA, ConA, and PWM stimulation; however, only a weak response to ConA and PWM was observed in B cell-enriched cultures. The mitogenic effects of PHA-P, ConA, and PWM on feline lymphocytes appeared to be due primarily to T-cell activation.     

Immunological changes during pregnancy in the viviparous lizard, Chalcides ocellatus

Splenic cells from pregnant and non-pregnant viviparous lizards (Chalcides ocellatus) were stimulated in vitro with the mitogens, concanavalin A (Con A), phytohemagglutinin (PHA) and Escherichia coli lipopolysaccharide (LPS). Cell cultures from pregnant animals were significantly less responsive to Con A and PHA than comparable cultures from non-pregnant animals. The response was depressed during the first period of pregnancy and remained low in magnitude until parturition. By contrast, the response of maternal splenic cells to LPS was reduced in pregnant lizards only during advanced pregnancy. The drastic decrease in mitogenic responsiveness was associated with marked involution of the maternal spleen. These findings strongly suggest that pregnancy impairs the immunoreactivity of viviparous lizards. Possible mechanisms for this impairment and the relationship to circulating levels of sex hormones are discussed.     

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.     

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.     

Nylon wool column fractionation and characterisation of feline lymphocyte subpopulations

Feline separated mononuclear cells (SMC) were obtained from peripheral blood by ficoll-diatrizoate gradient separation. SMC were further fractionated on nylon wool columns into nylon wool adherent cells (NWAC) and nylon wool effluent cells (NWEC). The three cell populations, SMC, NWAC and NWEC, were characterised using direct immunofluorescent staining for surface immunoglobulin (sIg) as a B cell marker and neuramidase treated guinea pig erythrocyte-rosette formation (E-rosettes) and mitogen-induced lymphocyte blastogenesis (LB) as possible T-cell markers. Feline SMC consisted of 30.1 +/- 4.0% sIg+ cells 36.6 + 5.4% E-rosette forming cells and 33.3% null cells i.e. cells which were sIg- and non E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming. Fractionation of SMC on nylon wool columns yielded NWEC which were significantly enriched for T cells in that they contained 68.6 +/- 2.9% E-rosette forming cells. NWAC were 51.0% +/- 10.8% sIg+, approximately 20% of cells were lost. The LB responsiveness of NWEC to concanavalin A (Con A) and phytonaemagglutinin-P (PHA-P) was enhanced compared to SMC. NWAC were non-responsive to Con A and PHA-P at all concentrations tested. It was concluded that nylon wool column fractionation of feline SMC was an efficient procedure for T cell enrichment and that the enriched cells retained the properties of E-rosette formation and blastogenesis by mitogens.     

A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes

A simple and reproducible micromethod for determination of in vitro mitogenic responses of sheep peripheral blood lymphocytes is described. The test uses (i) whole blood diluted in RPMI 1640 medium to give a cell count of 0.5 x 106-1 x 106 lymphocytes/ml, (ii) mitogens in the range of 5-20 micrograms of phytohaemagglutinin/ml, 20-80 micrograms of lipopolysaccharide/ml or 20-80 microliters of poke weed mitogen/ml, and (iii) a stimulation time of 42 to 90 h. A considerable variation in mitogenic response was observed both between animals and on different occasions in the same animal. Because of the large periodic variation it was suggested that the test should be repeated using blood drawn at different times in order to determine the mitogenic response of an animal.     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

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).     

Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes

The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied. Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2. The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action. Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM. The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM. The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum. When proliferation kinetics were studied, the peak response was observed on Day 2.     

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

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

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.     

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.     

Mitogen stimulation of canine normal and myasthenia gravis lymphocytes

Responses of canine lymphoid tissues to mitogens were studied in five normal dogs and in two dogs with acquired myasthenia gravis (MG). In the normal dogs, lymph-node-derived lymphocytes gave the most consistent proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), as determined by thymidine incorporation; and in most cases PHA, lipopolysaccharide (LPS), and PWM stimulated total IgG production, as determined by ELISA. Splenic lymphocytes had the greatest capacity for increased total IgG production. In the myasthenic dogs total IgG production by unstimulated lymph-node-derived lymphocytes was 88 micrograms/ml and 153 micrograms/ml, much higher than that of unstimulated normal dog lymphocytes (mean less than 1.0 microgram/ml). All mitogens resulted in suppression rather than stimulation of IgG production by lymphocytes from dogs with MG. Production of antibodies to acetylcholine receptors (AChRs) was detected in the supernatants of lymphocyte cultures from one of the dogs with MG at a rate of 78 fmol/5 x 10(5) cells per week and was not detected in culture supernatants of control dogs. This study demonstrates that lymph nodes may be an important site of antibody production in myasthenic dogs and provides the necessary groundwork for future studies of the cellular immunology of canine MG.     

Duck lymphocytes. VI. Requirement for phagocytic and adherent cells in lymphocyte transformation.

Preparations of duck (Anas platyrhynchos) spleen and blood lymphocytes depleted of cells capable of phagocytosing carbonyl iron gave lower transformation responses to the mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), lentil lectin (LL) and phorbol ester (PMA) than intact cell preparations. When cell populations were fractionated on the basis of their adherence to plastic, it was found that the adherent cells were responsive to PHA, Con A, BSS, WGA and PMA, while the non-adherent cells responded to LL. These observations confirm the expected requirement for phagocytic accessory cells in the induction of in vitro mitogen-driven duck lymphocyte responses. The responses of plastic-adherent populations of cells to most mitogens are believed to reflect the generally close physical relationship between the adherent accessory cells and the lymphocytes, although it remains possible that duck monocytes respond to some of the mitogens employed. The data also suggest that LL stimulates a population of cells different to those responding to other mitogens.     

A technique for inducing B-cell ablation in chickens by in ovo injection of cyclophosphamide.

The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of incubation. Blood samples were collected periodically from CY-treated and nontreated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 wk of age and booster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40%, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, B-cell responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by hemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. This study indicates the importance of monitoring the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.     

Starvation-induced pathobiology in the gut of carp (Cyprinus carpio L.)

In order to assess the effects of starvation on intestinal intraepithelial lymphocytes (i-IEL) count and histological changes of the carp gut mucosa, one group of fish (n = 10) were fed commercially prepared standard diets and another group of fish (n = 10) were starved for 4 weeks. Carp starved for 4 weeks developed enteropathy, comprising folds atrophy, stratum compactum hyperplasia, significant periodic-acid-Schiff (PAS)-positive (P < 0.00001), but not Alcian blue (ALB)-positive, goblet cell (GC) hyperplasia and a significant decrease (P < 0.00001) in i-IEL numbers. These changes were associated with a dense cellular infiltrate into the lamina propria. Taken together, these data suggest that the pathobiology of starvation-induced i-IELs decrease, matching PAS-positive goblet cell proliferation and inflammatory cells homing to the gut, could be classified as a non-infectious enteropathy induced by starvation.     

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.