Lymphocyte Blastogenic Response Research Articles

Strain differences in lymphocyte responses and in vitro suppressor cell induction between Schistosoma mansoni-infected C57BL/6 and CBA mice.

Splenic lymphocyte blastogenic responses to mitogens and antigens were compared in Schistosoma mansoni-infected C57BL/6 and CBA mice. At 1 and 2 weeks of infection, elevated responses to phytohemagglutinin (PHA) were noted in C57BL/6, but not in CBA cultures. During the same period, elevated responses to lipopolysaccharide were observed in cultures from CBA, but not from C57BL/6 mice. Responses to both mitogens were depressed in both strains later in infection. The magnitude of antigen-specific responses was greater in CBA than in C57BL/6 cultures. A cercarial extract (CE) induced significant suppressor cell activity in normal C57BL/6 cell cultures as assessed by inhibition of syngeneic cell responsiveness to PHA. CE did not induce suppressive activity in CBA cultures. CE-induced suppression was unaffected by adherent cell removal, but was sensitive to anti-Thy 1.2 plus complement treatment. CE also directly inhibited PHA-stimulated C57BL/6 cell cultures, but enhanced cultures of CBA mice. The significance of these findings is discussed in relation to the relative strain differences seen in antigen-specific lymphocyte proliferation and the levels of protection induced after immunization with irradiated cercariae.

Clinical studies on cell-mediated immunity in gestational trophoblastic disease: nonspecific cellular immunocompetence in patients with hydatid mole and trophoblastic neoplasia.

The nonspecific cell-mediated immunocompetence of 51 patients with gestational trophoblastic disease (GTD), including 16 patients with hydatid mole (HM), 24 with nonmetastatic trophoblastic neoplasia (NTN) and 15 with metastatic TN (MTN), was studied with the use of both in vitro and in vivo parameters of cell-mediated immunity (CMI) such as lymphocyte blastogenic response to phytohemagglutinin (PHA), subpopulation constitution, and delayed cutaneous hypersensitivity responses to 2,4-dinitrochlorobenzene (DNCB) and to purified protein derivative of tuberculin (PPD). The pretreatment cell-mediated immune status of patients with HM developing no malignant sequelae was shown to be essentially similar, in terms of both in vitro PHA and in vivo DNCB reactivities, to that of normal women and of patients with benign gynecological diseases. In patients with TN, however, there was a significant depression in the blastogenic lymphocyte response to PHA before evacuation of the mole, which was persistently demonstrated after uterine evacuation and more marked throughout the course of disease in patients with MTN, than in those with NTN, with a tendency to return to normal in remission. Moreover, patients with TN had a significant depletion of T lymphocytes as determined by rosette-forming cell procedures before treatment, which was most evident in patients with MTN. Plasma from the MTN patients was also shown to have an inhibitory effect on PHA responsiveness of lymphocytes from normal women. There was an increased incidence of impaired reactivity to DNCB in patients with TN (higher in MTN than in NTN), compared with HM and benign diseases, while no such difference in incidence was observed in response to PPD. On the basis of these findings, a preliminary characterization of altered immunocompetence in patients with TN and its mechanism are discussed.

Levamisole potentiation of antigen specific lymphocyte blastogenic response in [formula omitted][formula omitted] exposed but nonresponsive cattle

Incubation of Brucella abortus (field strain) infected and strain 19 vaccinated bovine peripheral blood lymphocytes with B. abortus antigen and levamisole caused a consistently significant increase in [ 3H] thymidine uptake when compared to cultures without levamisole. Levamisole did not potentiate B. abortus -induced blastogenic response of lymphocytes from non-exposed cattle. A dose response study showed that 10 μg/culture induced maximum potentiation of B. abortus -induced lymphocyte stimulation. Using the 10 μg/well concentration of levamisole, further studies were conducted to determine the net potentiation of the blastogenic responses in lymphocytes from B. abortus (field strain) infected cattle, B. abortus strain 19 vaccinated but nonresponsive and non-exposed cattle. Levamisole significantly potentiated the B. abortus -induced lymphocyte blastogenesis in lymphocytes from unresponsive cattle.

Effect of malnutrition and BCG vaccination on macrophage activation in guinea pigs

Host defense functions, particularly cell-mediated immunity, may be profoundly altered by malnutrition. Activated macrophages are required as effector cells in the final stages of a successful cell-mediated immune response against microbial pathogens. Macrophage activation was assessed by measuring the activity of superoxide dismutase (SOD) in peritoneal macrophages obtained from BCG-vaccinated and non-vaccinated guinea pigs maintained on purified diets containing 8% or 25% casein. Protein deficiency, as measured by reduced hemoglobin and serum albumin concentrations, was accompanied by significant reductions in delayed hypersensitivity to purified protein derivative (PPD) and impaired lymphocyte blastogenic responses in vitro to PPD and phytohemagglutinin. In contrast, macrophage SOD activity was inversely correlated with protein nutritional status, irrespective of vaccination. These results suggest that the thymus-dependent (T) lymphocyte subsets responsible for delayed hypersensitivity, blastogenesis and macrophage activation may be affected differently by protein deprivation. Alternatively, enhanced macrophage activity may not be the result of T cell function in this model.

Effect of single oral dose of phenobarbitone on lymphocyte blastogenic response in man.

A single oral dose of phenobarbitone 1.5 mg kg-1 was given to 18 normal subjects to evaluate the effects on lymphocyte function. Serum barbiturate concentrations and lymphocyte blastogenic response to three mitogens (PHA, Con A, PWM) were tested before and at 2, 12 and 36 h after drug administration. A serum barbiturate plateau of about 2 microgram ml-1 was maintained from 2 to 36 h. Lymphocyte blastogenesis was reduced with barbiturate compared with controls. However, the reduction was significant only at 36 h (P < 0.01).

Host Defense Deficiency in Hairy Cell Leukemia and its Correction by Leukocyte Transfusion

A similar defect host defense mechanisms in hairy cell leukemia was defined in two patients. Surface-adherent monocytes were not detected in the peripheral blood nor were monocytes that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) to isoantibody-coated human erythrocytes. In addition, lymphocytes of both patients failed to show blastogenic responses to concanavalin A (Con-A) and pokeweed mitogen (PWM) but showed a vigorous response to phytohemagglutinin (PHA). Other immunologic abnormalities were present but were either moderate in degree or were not present in both patients. In vitro lymphocyte blastogenic responses were fully restored by incubation of patients' leukocytes with a normal donor's adherent monocytes. One patient received daily allogeneic leukocyte transfusion for 4 days. This resulted in complete normalization of monocyte adherence and ADCC that persisted for several months after transfusion and was associated with hemotalogic improvement. Therapy in case 1 resulted in correction of the blastogenic responses to Con-A and PWM. Thus, a host defense defect in hairy cell leukemia has been defined in 2 patients and a preliminary result suggests that therapy with leukocyte transfusions may be useful in the postsplenectomy patient with an infectious complication and should be explored further.

Immune Responses during Human Schistosomiasis Mansoni

The lymphocyte blastogenic responses of chronic schistosomiasis mansoni patients were tested in vitro in medium supplemented with either normal human serum or patients' serum (either autologous or third party). As expected, when patients' lymphocytes were cultured in patient sera, many of them (75--78%) displayed reduced responsiveness to schistosome antigens (derived from either the cercariae, adult worms or eggs of Schistosoma mansoni), but not to Candida albicans extract. For decreased blastogenesis to be manifest, a combination of both suppressive sera and suppressible cells was required; however, some patients had nonsuppressible cells and not all sera were suppressive. In an attempt at classification, four categories of patient responsiveness concerning serosuppression are proposed. The categories depend on the suppressive capabilities of patient sera and the response of patient lymphocytes to suppressive sera. By individually testing the capabilities of each patient's lymphocytes and sera in relationship to each antigenic preparation, we were able to assign the majority of patient responses to a given category. It is hoped that by using these categories, a better understanding of the mechanisms concerning serosuppression will be obtained.

Host defense deficiency in hairy cell leukemia and its correction by leukocyte transfusion

A similar defect host defense mechanisms in hairy cell leukemia was defined in two patients. Surface-adherent monocytes were not detected in the peripheral blood nor were monocytes that mediate antibody- dependent cell-mediated cytotoxicity (ADCC) to isoantibody-coated human erythrocytes. In addition, lymphocytes of both patients failed to show blastogenic responses to concanavalin A (Con-A) and pokeweed mitogen (PWM) but showed a vigorous response to phytohemagglutinin (PHA). Other immunologic abnormalities were present but were either moderate in degree or were not present in both patients. In vitro lymphocyte blastogenic responses were fully restored by incubation of patients' leukocytes with a normal donor's adherent monocytes. One patient received daily allogeneic leukocyte transfusion for 4 days. This resulted in complete normalization of monocyte adherence and ADCC that persisted for several months after transfusion and was associated with hemotalogic improvement. Therapy in case 1 resulted in correction of the blastogenic responses to Con-A and PWM. Thus, a host defense defect in hairy cell leukemia has been defined in 2 patients and a preliminary result suggests that therapy with leukocyte transfusions may be useful in the postsplenectomy patient with an infectious complication and should be explored further.

Lymphoproliferative responses to oral bacteria in humans with varying severities of periodontal disease.

We assessed in vitro the lymphocyte blastogenic resp onsess of peripheral blood lymphocytes to antigen extracts of a large battery of oral microorganisms in a population of humans with varying severities of periodontal disease. When the magnitudes and frequencies of statistically positive blastogenic responses to various oral microorganisms were compared, three general patterns emerged. The Actinomyces species proved to be potent stimulators of lymphocyte blastogenesis in most subjects tested, whereas Streptococcus sanguis, Campylobacter, and Eikenella corrodens stimulated few individuals. The response to these organisms correlated poorly with the severity of periodontal disease in the tested patients. However, several gram-negative anaerobic organisms, including Bacteroides asaccharolyticus and Treponema denticola, elicited statistically more frequent positive response in subjects with destructive periodontitis compared with patients with gingivitis. These results, taken together with recent microbiological findings, suggest that the specificity of the lymphocyte blastogenic response to antigens of oral bacteria correlates with the presence of these organisms in the subgingival microflora during various periodontal disease states.

Comparative in vitro imunotoxicology of acyclovir and other antiviral agents

In vitro lymphocyte blastogenic responses to the commonly employed mitogens phytohemagglutinin, pokeweed, and concanavalin A were evaluated when acyclovir, adenine arabinoside, cytosine arabinoside, and idoxuridine were added to the culture materials. Similarly, specific antigen-induced blastogenic responses, including herpes group antigens, and cytotoxicity and leukocyte inhibitory factor assays with herpes group viruses were determined in the presence and absence of antiviral agents. No depression of these cellular immmune responses by acyclovir or adenine arabinoside ws demonstrated. This was in contrast to the effects of cytosine arabinoside and idoxuridine, which severely inhibited blastogenic and cytotoxic responses but not leukocyte inhibitory factor production. Even at concentrations up to 20 microgram/ml, the antiviral agent acyclovir did not depress selected cellular immune responses that are important for successful elimination of invading herpes group viruses.

The influence of mitogen costimulation on the human lymphocyte blastogenic response to Actinomyces viscosus ultrasonicates

Human peripheral blood lymphocytes were cultured for 48 hr in the presence of an ultrasonicate supernatant fraction of Actinomyces viscosus (AV-SUP). After washing to remove AV-SUP, the lymphocytes were cultured in the presence of concanavalin A or pokeweed mitogen for an additional 72 hr. Measurement of [ 3H]thymidine incorporation into DNA indicated that AV-SUP preculture resulted in an amplified or elevated response to mitogen stimulation compared to mock precultured cells and unstimulated controls. Addition of fresh cells prior to mitogen activation resulted in an additional incremental increase over controls. The modulated cell appeared to reside in the AV-SUP precultured population, since treatment of these cells with mitomycin C prior to coculture with fresh cells totally abrogated the effect. However, mitomycin C treatment of the fresh population had no effect on mitogen-induced amplification. Results of these experiments suggest that mitogen stimulation activates a helper function which allows the precultured cells to respond to AV-SUP stimulation.

Breast milk lymphocyte response to K1 antigen of Escherichia coli.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli O127,B8 were examined in 16 postpartum women by [3H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of greater than or equal to 2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.

Sequential evaluation of immunoreactivity in patients with melanoma undergoing surgery and adjuvant therapy.

The immunologic profile of 15 patients undergoing surgery and adjuvant chemoimmunotherapy for cutaneous melanoma was studied for a mean period of 18 months. In vivo cellular immunity was assayed by evaluation of delayed hypersensitivity response (DHR) to primary antigen and a panel of recall antigens. In vitro cellular immunity was evaluated to means of total and T-lymphocyte counts in peripheral blood and by the lymphocyte blastogenic response to phytohemagglutinin stimulation. Humoral immunity was assayed by determining the serum levels of IgG, IgA and IgM and of complement components C3c, C4 and Factor B. Phagocytic activity was studied by testing leukocyte chemotaxis, neutrophil phagocytosis and leukocyte random migration. The in vitro parameters were determined preoperatively at diagnosis, 6 times during the first 2 postoperative weeks, and then every month during adjuvant therapy. No correlation was found between DHR and clinico-pathologic stage of tumor, or with subsequent clinical course. Significant depression of total lymphocyte and T-lymphocyte count and blastogenic response of lymphocytes was found at diagnosis. The lymphocyte response to PHA decreased significantly in the early postoperative period but returned to preoperative levels one week after surgery. Periodic fluctuations of lymphocyte blastogenic response and progressive decrease of total lymphocyte counts and T-lymphocyte counts were observed during the 18-month follow-up. No significant alterations of immunoglobulin levels were recorded at diagnosis or during the postoperative period. Complement levels were within normal values preoperatively; in the early postoperative period a transient increase of C3c, C4 and Factor B was recorded, then complement levels progressively decreased. Parameters of phagocytic activity were normal at diagnosis and fluctuated within the normal range throughout the whole period of study.

The influence of incubation time and mitogen concentration on lymphocyte blastogenic response: determination of conditions that maximize population differences.

The blastogenic responsiveness of peripheral blood lymphocytes is a commonly applied measure of immune response capability. This assay is generally run under conditions of maximum lymphocyte stimulation. The purpose of this study was to evaluate which conditions of lymphocyte culture were able to best discriminate normal from subnormal lymphocyte responses to mitogens and antigens. Mitogen or antigen concentrations and the length of incubation time were varied while other assay conditions (serum supplement, lymphocyte concentration, amount of label, and length of label pulse) were kept constant. A total of 24 cancer patients and 25 normal controls were studied. Under optimal assay conditions that provided for maximum lymphocyte stimulation, we were unable to distinguish differences in response between young and old individuals or cancer patients and normal controls. However, at suboptimal mitogen/antigen concentrations or suboptimal incubation times, significant differences were observed in these populations. It is suggested that in the evaluation of lymphocyte stimulation responses in various clinical groups, suboptimal lymphocyte culture conditions may provide for maximum discrimination among the groups.

Potential use of lymphocyte blastogenic responses in diagnosis of bovine tuberculosis

A comparison of in vitro lymphocyte responses and delayed type tuberculin skin test responses was made in an animal experimentally exposed to a Mycobacterium bovis-infected animal and in cattle naturally infected with M. bovis. Tuberculin skin tests did not suppress in vitro lymphocyte responses to M. bovis PPD and to M. avium PPD tuberculin. The whole blood test used in these studies provided for considerable savings in time as compared to use of purified lymphocytes for evaluating in vitro cellular responses. Variations in the responsiveness of lymphocytes to specific mycobacterial antigens was observed, therefore, it is recommended that profiles be established using three or more tests conducted at 14-day intervals.

Lymphocyte Blastogenic Response to Autologous Tumor Cells Pretreated with Neuraminidase in Patients with Gastric Carcinoma

The effect of Vibrio cholerae neuraminidase (VCN) treatment of tumor cells on lymphoblastic response was studied using the assay of mixed lymphocyte-tumor cell reaction in 24 patients with gastric carcinoma. Lymphoblastic responses as indicated by stimulation index were significantly augmented in cultures with autologous tumor cells pretreated with mitomycin (MMC) and VCN as compared to those pretreated with MMC alone when cultured in the presence of normal plasma (p < 0.05). In autologous plasma cultures, however, the mean stimulation index in MMC-VCN-treated cell cultures was only slightly higher than that in MMC-treated cell cultures. Accentuation of the response was not observed in any type of cultures using MMC-VCN-treated normal tissue cells. In 20 of these patients, the blastic response to nonviable frozen stored tumor cells was measured simultaneously and significant increase in blastic transformation by pretreatment of the cells with VCN was also observed in normal plasma cultures (p < 0.05). There was significant correlation between the response to nonviable frozen VCN-treated cells and to these, not frozen, viable cells (p < 0.001). These results indicate that lymphoblastic response to autologous tumor cells could be enhanced by pretreating the cells with VCN in patients with gastric carcinoma and the freezing could render the cells nonviable without interfering with the augmented immunogenicity of VCN-treated tumor cells.

The lymphoproliferative response during human experimental gingivitis

The lymphocyte blastogenic responses to antigen preparations of bacteria from dental microbial plaque and to other non‐oral stimulants were longitudinally assayed in eitht adult subjects using the experimental gingivitis model. During the 28 days of the experimental period, these subjects abstained from all oral hygiene procedures which let to accumulatio of increased amounts of microbial dental plaque and subsequent gingivitis. In general, all subjects demonstrated a significant increase in the lymphoproliferative response to all doses of phytohemagglutinin (PHA) during the experimental period. The subjects were divided into 2 groups based on rate of plaque accumulation. Slow plaque formers exhibited a gradual linear increase in blastogenic response to bacterial antigen preparations throughout the experimental period. In the group characterixed by rapid accumulation of plaque, a marked early increase in blastogenic responses to antigens of Actinomyces viscosus, Actinomyces naeslundii, Bacteroides melaninogenicus, and Treponema denticola was observed. These same subjects, however, demonstrated a reduction in lymphocyte response to the above antigens during the later phase of the experimental period, when gingivitis was most severe. This decrease, in light of the similtaneous increase in PHA response, suggests the existence of a homeostatic mechanism to control the immune response during episodes of increased antigen load.

Initiation of the blastogenic response of lymphocytes by hyperoptimal concentrations of concanavalin A.

The blastogenic response of human lymphocytes in vitro to hyperoptimal concentrations of concanavalin A (Con A) has been studied by means of volume spectroscopy (measuring cellular and nuclear volume), flow cytofluorometry (measuring cellular DNA content) and incorporation of [3H]thymidine ([3H]dThd). The optimal Con A dose with respect to [3H]dThd incorporation was about 30 micrograms/ml. In cultures given hyperoptimal doses, e.g. 100 micrograms/ml, [3H]dThd incorporation was strongly inhibited, whereas the number of cells entering S-phase and significantly increasing their cellular and nuclear volume was considerably larger than with 30 micrograms/ml. With 200 micrograms/ml Con A, which induced negligible [3H]dThd incorporation, the percentage of responding cells was even larger. Hence, doses of Con A, which were hyperoptimal with regard to [3H]dThd incorporation, induced blastogenic response, including DNA synthesis, in a larger percentage of the cells than did the optimal dose. However, in cultures with hyperoptimal Con A doses, the progression of the cell cycle stagnated mainly during S- and G2-phase and few cells completed mitosis. Thus, the blocking effect of hyperoptimal doses was not confined to any particular point of the cell cycle. The reduced [3H]dTd incorporation, seen with hyperoptimal doses, is attributed partly to a failure of this assay under such conditions.

Partial Purification and Characterization of Schistosoma Mansoni Soluble Egg Antigen with Con A-Sepharose Chromatography

Abstract A crude preparation of Schistosoma mansoni soluble egg antigen (SEA) was subjected to affinity chromatography with concanavalin A (Con A) bound to Sepharose 4B. The resulting Con A fractions (bound and unbound) were characterized with sodium dodecyl sulfate (SDS) gel electrophoresis, immunoelectrophoresis, immunodiffusion, and lymphocyte blastogenesis techniques. In the fraction that did not bind to Con A there were at least two distinct antigens, and there were also at least two distinct antigens in the fractions that did bind to Con A. With SDS polyacrylamide gel electrophoresis, at least 20 distinct protein bands (Coomassie blue staining) and three glycoprotein bands (PAS reactive) were present in the unbound fractions from Con A chromatography. The bound fractions separated into at least six distinct glycoproteins with SDS electrophoresis. Although both the bound and unbound fractions contained precipitating antigens, only the bound fractions were capable of eliciting lymphocyte blastogenic responses.

The effect of prostaglandin E1 or E2 on the in vitro blastogenic response of lymphocytes from normal and tumor-bearing mice.

The in vitro effect of exogenously added prostaglandin (PG) E1 or E2 over concentrations ranges of from 1 X 10(-4) to 1 X 10(-9) M were studied in order to determine their effect on the in vitro lymphocyte proliferation of thymic and splenic T and B cells from normal and tumor-bearing CD2F1 mice. It was found that PGE1 generally caused greater inhibition of blastogenesis than did PGE2 when reacted with splenic lymphocytes from normal mice. Indeed, PGE2 was found to be stimulatory for both Con A- and LPS-sensitive normal splenic lymphocytes. Both PGE1 and PGE2 caused potent inhibition of Con A- and PHA-sensitive splenic lymphocytes from the tumor-bearing mice. Additionally, PGE2 was found to stimulate the LPS-reactive lymphocytes from the tumored mice. PGE1 and PGE2 both inhibited the Con A- and PHA-reactive thymic lymphocytes from normal mice at the lower concentrations studied, i.e., 10(-4) to 10(-6) M. Thereafter, at concentration ranges of from 10(-7) to 10(-9) M both PGE1 and PGE2 were both found to be stimulatory. Finally, both PGE1 and PGE2 at all concentrations studied, strongly inhibited the thymic lymphocytes from tumored mice.