Inhibition of lymphocyte blastogenesis by whey

Bovine whey samples were evaluated by use of lymphocyte-transformation tests to determine their effect on lymphocyte blastogenesis. Whey samples from mammary glands with clinical mastitis strongly inhibited DNA synthesis and blastogenesis in lymphocytes stimulated with mitogens or dividing because of bovine leukemia virus infection. Whey samples from apparently healthy glands either did not inhibit lymphocyte DNA synthesis or inhibited it to a lesser degree than did whey from mastitic glands. Degree of inhibition was dose-dependent. The molecules causing inhibition were noncytotoxic and underwent minimal binding to the lymphocytes. Inhibitory molecules were susceptible to various proteolytic and glycolytic enzymes, indicating a glycoprotein-like structure. Whey inhibited incorporation of thymidine if it was in the cell cultures during the early stages of stimulation. Incubation of lymphocytes in whey that inhibited thymidine incorporation did not affect DNA synthesis in subsequent culturing of the same cells without whey. Degree of inhibition was affected by the method of whey preparation.     

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.     

Inhibition of in vitro lymphocyte blastogenesis by chicken alpha-fetoprotein

Chicken alpha-fetoprotein (ch-AFP), purified from fetal chicken serum and embryo extracts, respectively, was examined for its immunomodulatory effect in vitro. Significant (P less than 0.05) suppression of the allogeneic mixed lymphocyte reaction (MLR) was observed, when these preparations were added to one-way mixed lymphocyte cultures (MLC) in quantities of 62.5-1000 micrograms/ml. Suppression of the MLR was depending on the presence of ch-AFP for at least 16 h after initiation of the MLC, suggesting that this fetal protein was acting mainly in the early phase of lymphoblastogenesis. Serum of chicken embryos (12th and 15th day of incubation), day-old chickens, and of 10-week-old chickens of four different inbred lines were also found to exert suppression of the MLR. From these data, it is hypothesized that ch-AFP plays an immunoregulatory role by maintaining a certain stage of self tolerance during differentiation of the avian immune system.     

Suppression of lymphocyte blastogenesis by porcine interferon-α

Peripheral blood lymphocytes (PBL) from five four-day-old and five six-week-old piglets were treated with 10 to 320 units of porcine interferon-alpha, and their blastogenic responses to phytohaemagglutinin or pokeweed mitogen were compared with those of control lymphocytes. There was significant inhibition of the blastogenic response to phytohaemagglutinin by 320 units of interferon-alpha, and of the response to pokeweed mitogen by 320 and 160 units of interferon-alpha. Porcine interferon-beta was cytotoxic to porcine PBL. The blastogenic response to pokeweed mitogen was significantly higher in PBL from the younger piglets.     

Suppression of lymphocyte blastogenesis by sera from calves transported by road

The effect of sera from 4-h road-transported calves on mitogen-induced blastogenesis of bovine lymphocytes was investigated. Sera collected just after transportation showed a significant suppression (P less than 0.05) on blastogenesis. The immunosuppressive activity was reduced from those sera when they were treated with dextran-coated charcoal. Fractionation of the sera by ultrafiltration revealed that the activity was mainly concentrated in the fractions of molecular weights less than 50 kD.     

Effect of levamisole on lymphocyte blastogenesis and neutrophil function in dexamethasone-treated cattle

Levamisole was evaluated at 6 dose levels for its ability to prevent the dexamethasone-induced suppression of in vitro lymphocyte blastogenesis or neutrophil function in cattle. Dexamethasone (0.4 mg/kg of body weight, IM) and levamisole hydrochloride (0.5, 1.0, 2.0, 4.0, or 8.0 mg/kg orally) were administered to groups of 4 cattle daily for 3 days. Another group of 4 cattle were given the 3-day dexamethasone treatment and 6.0 mg/kg of levamisole (the recommended anthelmintic dose) was given only once on the 1st day that dexamethasone was given. Results obtained from the dexamethasone-levamisole-treated cattle were compared with results obtained from cattle that were given only dexamethasone. Levamisole had no apparent consistent ability to enhance lymphocyte blastogenic responsiveness (to the mitogens phytohemagglutinin, concanavalin A, or pokeweed mitogen or in a 1-way mixed lymphocyte reaction) or to enhance neutrophil function (random migration, nitroblue tetrazolium reduction, iodination, or antibody-dependent cell-mediated cytotoxicity) in dexamethasone-treated cattle.     

Effects of ACTH administration on bovine polymorphonuclear leukocyte function and lymphocyte blastogenesis

Yearling steers were treated with ACTH to determine the effect of increased plasma cortisol concentration on bovine lymphocyte and polymorphonuclear leukocyte (PMN) function. The administration of ACTH caused a significant (P less than 0.01) increase in serum cortisol concentration and depression of lymphocyte blastogenesis in response to phytohemagglutinin and concanavalin A. The response to pokeweed mitogen was also depressed, but not significantly. Random migration by PMN was significantly enhanced by ACTH treatment, but there was no effect on ingestion of Staphylococcus aureus, nitroblue tetrazolium reduction, or antibody-dependent cell-mediated cytotoxicity by PMN. The iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the PMN, was significantly impaired after ACTH treatment. These data indicate that specific parameters of lymphocyte and neutrophil function were impaired directly or indirectly by elevated in vivo concentrations of plasma cortisol.     

Effect of hydrocortisone on circulating lymphocyte numbers and their mitogen-induced blastogenesis in lambs

The effect of hydrocortisone on the number of circulating lymphocytes and their blastogenic response was studied in 20 feedlot lambs given combinations of 3 treatments: hydrocortisone (25 mg/kg of body weight, 4 times a day, IM), feed changes (100% roughage to 90% concentrate over a 6-day period), and oral inoculation of Pasteurella haemolytica biotype T (10(9) to 10(11) bacteria/day via stomach tube) to develop a model for reproduction of septicemic pasteurellosis. Hydrocortisone caused lymphopenia and inhibited the blastogenic response of peripheral blood lymphocytes to phytohemagglutinin and concanavalin A mitogens. A synergistic effect was observed between hydrocortisone injections and feed changes resulting in higher than expected serum hydrocortisone concentrations and lower circulating lymphocyte counts. Seemingly, stress-induced increases in serum hydrocortisone concentrations cause suppression of the immune response of feedlot lambs. The combined effect of feed changes and stress on the immune response of lambs may explain the role of these 2 factors in the pathogenesis of septicemic pasteurellosis.     

Alterations in neutrophil phagocytosis and lymphocyte blastogenesis in dairy cows around parturition

The bovine blood neutrophil phagocytosis and the blood and milk lymphocyte proliferative response upon stimulation with Phytohaemagglutinin, Concanavalin A and Pokeweed mitogens was studied from 3 weeks prior to calving until 3 weeks after calving. Neutrophil phagocytosis and the total and differential blood leukocyte counts were performed by flow cytometry. A gradual increase in the percentage of phagocytized bacteria and the average number of bacteria per phagocyte was observed before calving followed by a sharp fall on the first postpartum. This was followed by a steady increase in the above parameters reaching the highest levels at two weeks postpartum. There was a gradual increase in the number of neutrophils in blood as calving approached followed by a sharp decrease after calving. The number of lymphocytes in blood dropped before calving, being at the lowest level on the day before calving. The proliferative response of blood and milk lymphocytes upon stimulation with the three mitogens was low during the week preceding parturition with the lowest value on the day before calving. The response of blood lymphocytes returned to a higher level the second week after calving while that of milk lymphocytes remained at a low level during the first and the second postpartum weeks.     

Immune response of sheep to bluetongue virus: in vitro induced lymphocyte blastogenesis

Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance.     

Simple technique for examining lymphocyte blastogenesis in whole blood cultures for neonatal calves

A method for examining lymphocyte blastogenesis in whole blood cultures of neonatal calves is presented. Considerable variation in magnitude of thymidine incorporation was noted between animals but the general trend of response was uniform. Maximum responses occurred at different culture times for each mitogen. In a standard 0.2 ml micro culture system using 10,000 mononuclear cells per culture incubated for 96 hours in 5 per cent fetal bovine serum maximum mitogenic responses were obtained with 0.8 microgram phytohaemagglutinin per culture, 1 microgram concanavalin A per culture and 0.08 microgram pokeweed mitogen per culture.     

Repeated suppression of lymphocyte blastogenesis following vaccinations of CPV-immune dogs with modified-live CPV vaccines

A commercially available modified-live canine parvovirus (CPV) vaccine was evaluated for its immunosuppressive properties in eight random-bred dogs, all with circulatory antibody to CPV. Three of the eight dogs exhibited a significant decrease in lymphocyte blastogenesis after vaccine administration. In these dogs, this decrease in blastogenesis was of short duration and was consistently observed after repeated administrations of the vaccine. Neither gastroenteritis, fever nor leukopenia, signs indicative of virulent canine parvovirus infection, were detected in these animals. In addition, lymphocytes from these dogs lacked Ia antigen expression. This study demonstrated that the immunomodulating effects of ML-CPV is not observed in all animals yet is consistent in affected individuals.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.     

Enhancement of lymphocyte blastogenesis and neutrophil function by avridine in dexamethasone-treated and nontreated cattle

The lipoidal amine, N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine (avridine or CP 20,961), formulated in liposomes, was evaluated for its effect on leukocyte kinetics, lymphocyte blastogenesis, and polymorphonuclear leukocyte (PMN) function in dexamethasone-treated and nontreated cattle. In the 1st experiment, cattle were given avridine in a single IM injection of 0.1, 1.0, or 10 mg/kg of body weight. All doses induced swelling at the injection site, a febrile response, and a leukocytosis due to a neutrophilia. Mononuclear cell numbers were normal. All 3 groups of avridine-treated animals had a higher mean lymphocyte blastogenic response to mitogens on the 4 days after administration than did the control nontreated animals. Avridine administration was associated with an enhanced ability of PMN to ingest Staphylococcus aureus and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The highest dose (10 mg/kg) was associated with a depression of the ability of PMN to iodinate protein. An effect of avridine on PMN random migration under agarose or nitroblue tetrazolium (NBT) reduction was not observed. In a 2nd experiment, cattle were given no treatment, 0.04 mg of dexamethasone/kg IM, or 10 mg of avridine/kg IM followed 24 hours later by 0.04 mg of dexamethasone/kg. Dexamethasone administration caused a leukocytosis due to a neutrophilia with normal mononuclear cell numbers, an enhancement of PMN random migration under agarose, and an inhibition of NBT reduction, iodination, and ADCC activity of PMN. Dexamethasone did not have a detectable effect on lymphocyte blastogenesis or on ingestion of S aureus by PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune response of cattle to Brucella abortus outer membrane proteins measured by lymphocyte blastogenesis

Lymphocytes from cattle were tested in a blastogenesis test with outer membrane proteins isolated from smooth strain 2308 and rough strain 45/20 of Brucella abortus. The titration assay developed for measuring blastogenesis to microbial antigens (Baldwin, Antczak and Winter, this issue, pp. 319-333) was used to assess the response to both group 2 (porins) (Douglas et al., 1984) and group 3 proteins (Verstreate et al., 1982). Blastogenesis was evaluated for distinguishing cattle infected with virulent B. abortus strain 2308 from unimmunized cattle, cattle vaccinated with attenuated strain 19, or inoculated with Escherichia coli 0116:H31, known to cause serological cross-reactions with B. abortus (Nielsen et al., 1980). Strain 45/20 porin was the most effective for this purpose and data analyses utilizing the titration assay were better than those relying on a single point assay. When compared with BASA, an antigen preparation used in other studies (Kaneene et al., 1978a), responses to porin provided a more specific index of infection with B. abortus. Reactions to 45/20 porin occurred, however, in some heifers vaccinated as adults with strain 19 or inoculated with E. coli 0116:H31. Furthermore, nonpregnant heifers had negligible or only transient blastogenesis responses to the porin during the first 14 weeks after infection even though they developed strong 0 antibody responses. We do not recommend the blastogenesis test in its present form as a useful adjunct to serological tests, and could allow measurement of cell mediated immune responses relevant to protective immunity.     

Evaluation of azathioprine on lesion severity and lymphocyte blastogenesis in dogs with perianal fistulas

Fourteen dogs with perianal fistulas were entered into a prospective clinical study to investigate the effects of long-term azathioprine on clinical outcome and to determine if the clinical results correlated with lymphocyte blastogenesis tests. Complete remission of perianal fistulas was seen in eight (57%) of 14 dogs; partial remission occurred in one (7%) dog; and no response was detected in five (36%) dogs. The results of lymphocyte blastogenesis assays did not correlate with therapeutic response.     

Suppression of specific lymphocyte blastogenesis after intravenous tuberculin injection in Mycobacterium bovis-sensitized cattle.

Intravenous injection of 1.0 ml of old tuberculin (OT) in 4 calves previously sensitized to Mycobacterium bovis resulted in suppression of specific lymphocyte blastogenic responses to purified protein in vitro. Suppression of in vitro blastogenic responses occurred within 24 hours after injection of OT and persisted for up to 96 hours. Lymphocytes from these calves, cultured with concanavalin A, did not show any suppression of the lymphocyte blastogenic response in vitro. Suppression was temporary and in all calves was preceded by rapid febrile responses. Rectal temperature responses ranged from 40.1 to 41.6 C as early as 6 hours after tuberculin injections. Three control animals, M bovis-sensitized but not given OT, remained lymphocyte stimulation-positive throughout the study and served as controls for laboratory fluctuations.