The effect of the seasonal cycle on the splenic leukocyte functions in the turtle Mauremys caspica

The reptile immune system is strikingly affected by seasonal variations, which induce changes in the structure of the lymphoid organs and in the function of the leukocytes. The aim of this work is to study several functions of splenic leukocytes from the turtle Mauremys caspica along its seasonal cycle. The functions assayed were adherence to substrate, mobility directed to a chemoattractant gradient (chemotaxis), lymphoproliferative response to mitogens, antibody-dependent cellular cytotoxicity, and natural killer-like cell-mediated cytotoxicity. Splenic leukocytes showed a positive response in all the assays, and this response was similar to that of mammals. In regard to the effect of the seasonal cycle, we have observed in winter a low adherence to substratum and high chemotaxis and cytotoxic activity, whereas in spring, only lymphoproliferation induced by mitogens showed high values except with lipopolysaccharide, which did not induce any seasonal variation in proliferation percentages. In summer, a high chemotaxis and cytotoxicity were observed, while in autumn, adherence to substratum was increased, but chemotaxis, cytotoxicity, and proliferation were clearly diminished. Our results demonstrate that splenic leukocyte functions are affected by the seasonal cycle, which induces a different pattern of response depending on the function studied.     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

The effect of a peat-based preparation on mitogen-induced proliferation of thymocytes in non-treated and hydrocortisone-suppressed mice.

The studies were carried out on Balb/c mice (5-6 weeks of age) treated with a peat-based preparation (PBP), administered i.p. once or four times at 24 h intervals at doses of 0.01; 0.1 or 1 mg/kg. Additionally, hydrocortisone was injected i.p. to selected mice at a single dose of 125 mg/kg. The results show that PBP temporarily enhances the proliferative capability of murine thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The effect of PBP depends on the number of subsequent doses, but does not depend on the dose applied. A single PBP administration does not affect the proliferative response of thymocytes to Con A and PHA. A single injection of PBP (doses from 0.01 to 1 mg/kg) does not change the number of thymic cells and weight ratio of this organ. Increased doses of subsequent PBP injections (0.01 and 0.1 mg/kg) do not affect the number of thymocytes, but temporarily increase the weight ratio of the thymus two days after the last injection. Administration of PBP prior to hydrocortisone prevents the suppressive effect of the drug on proliferative response of thymocytes stimulated in vitro with Con A and PHA, at the same time increasing the proliferative response of thymic cells to the two mitogens in relation to the control group (hydrocortisone-free). The effect of a single dose of PBP depends on the dose applied--the weakest preventive effect was observed at a dose of 0.01 mg/kg. An increase in the number of subsequent PBP doses, irrespective of a dose applied, prolongs the protective action of the drug on proliferative activity of thymocytes stimulated in vitro with these mitogenes. Moreover, the results obtained in the studies show that PBP partially prevents the suppressive effect of hydrocortisone, as the number of thymic cells and weight ratio of this organ drastically decreased. PBP accelerates regeneration of the thymus, but this depends on a dose applied and the number of subsequent doses. The result was the strongest and the fastest when PBP was injected four times at a dose of 1 mg/kg. It seems quite likely that the thymic regeneration due to PBP is connected with the effect of this drug on maturation and differentiation of thymic cells.     

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli. The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.     

Selective abrogation of alloreactivity via priming in the presence of aphidicolin, a specific inhibitor of DNA polymerase

Aphidicolin, a specific and direct inhibitor of eukaryotic DNA polymerase alpha, was used to investigate its impact on immunologic reactions in vitro. Dose response curve of the inhibitory effect was studied in murine and human primary allogeneic responses, as well as the proliferative responses to both PHA and Con A mitogens. The presence of aphidicolin during the allosensitization phase in secondary MLR of mice splenocytes resulted in complete abolishment of the subsequent response directed against the priming alloantigens, whereas alloreactivity to unrelated alloantigen-bearing cells was inhibited to a much lesser degree. The allosensitized aphidicolin-treated cells lost the ability to respond to subsequent PHA stimulation, but were capable of exerting a high responsiveness to Con A. The presence of aphidicolin during the allosensitization phase in secondary MLR of human mononuclear cells resulted in markedly decreased alloreactivity directed against the priming cells, but spared the subsequent response to unrelated alloantigens and to both PHA and Con A mitogenic stimuli. It is suggested that aphidicolin may be used for selective inactivation of proliferating cells without interfering with immunologic functions of other quiescent subsets. Aphidicolin may thus be a useful agent for induction of specific unresponsiveness in experimental models of allogeneic transplantation.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

The effect of a high external temperature on cellular immunity]

The study was made of spleen cells proliferative response to mitogens PHA, Con A or alloantigens in relation to hyperthermia effects. Acute hyperthermia (rectal temperature 42 degrees) enhanced lymphocyte function, proliferative responses to allo-antigens, PHA and Con A increased. Thermal shock was associated with suppression of the spleen cell response. Mice suffering from hyperthermia for 20 min (43-44 degrees) daily during 10, 20 and 30 days showed suppressed T-cell immune response. Normal splenocyte proliferation recovered 40 days after hyperthermia induction.     

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Differential suppressive effects of antiserum to thymus ribosomal fraction on mitogen responsiveness of thymus cells

The effect of antiserum to thymus ribosomal fraction (ATRS) on the responsiveness of thymus cells to mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was investigated. ATRS preferentially suppressed the responsiveness of thymus cells to Con A whereas their PHA responsiveness remained unchanged. In contrast, thymus cells from mice treated with a single injection of cortisone acetate showed significant increases in thymidine uptake when stimulated by both PHA and Con A.     

A history of theories of acquired immunity

Alloimmune mouse spleen cells are capable of carrying out nonspecific cell-mediated cytolysis of syngeneic target cells when incubated in the presence of lectins such as Con A or PHA (lectin-dependent cell-mediated cytotoxicity). In the present study plant lectins from a variety of sources were examined for their ability to participate in alloimmune-LDCC. Reactivity was then compared to mitogenic activity and the ability to activate cytotoxic effector cells in vitro. Of the lectins tested only those reported to be T-cell mitogens were capable of participating in alloimmune-LDCC. Agglutinating but nonmitogenic lectins (e.g., WGA) or mitogens such as LPS or PWM failed to yield positive LDCC. Of the T-cell mitogens demonstrating positive reactivity in the alloimmune-LDCC assay, only a portion were able to generate cytolytic activity when incubated with normal spleen cells in vitro (Con A, GPA, lentil). Crude PHA, purified erythroagglutinin, or leukagglutinin failed to generate cytotoxic effector cells in this system even though these were mitogenic and demonstrated positive alloimmune-LDCC. The results suggest that T-cell mitogens interact with cytotoxic effector cells in a manner which specifically triggers cytolysis. The relationship of this interaction to other lymphocyte-lectin interactions is discussed.     

Differential requirement for accessory cells in polyclonal T-cell activation

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.     

Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.     

Role of A cells in the mitogenic stimulation of lymphocytes by Mycoplasma

The dependence of the blast transformation of lymphocytes in response to phytohemagglutinin (PHA), concanavalin A (Con A) and Mycoplasma arthritidis on the concentration of A-cells and the time of the introduction of M. arthritidis into the culture was studied. The level of blast transformation in response to PHA, Con A and M. arthritidis increased with the decrease of the concentration of A-cells in the culture. After the combined inoculation of the culture with M. arthritidis and PHA the resulting effect was higher than that induced by PHA alone and lower than the level of blast transformation in response to M. arthritidis at all A-cell concentrations under study. After the combined inoculation of M. arthritidis and Con A the summation of response was observed in cultures with a high concentration of A-cells, while in cultures with a low concentration of A-cells the resulting response was lower than that induced by any of these mitogens alone. The inoculation of the culture with M. arthritidis 24-48 hours after the cultivation of splenocytes with PHA and Con A was started led to the suppression of response to the mitogens. The suppression of response to PHA was most pronounced at the maximum concentration of A-cells, while the suppression of response to Con A reached its highest level when the concentration of A-cells was low. These data are in accord with the suggestion that M. arthritidis and PHA, as well as M. arthritidis and Con A, stimulated the overlapping subpopulations of lymphocytes in rats, the adhesive properties being most pronounced in the subpopulation of PHA- and M. arthritidis-positive lymphocytes.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of humoral immunity.

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.     

A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity.

The ability of mitogens to induce cytotoxic effector reactions in vitro has been studied to investigate basic mechanisms of cell mediated cytotoxicity. The type of mitogen, the source of effector cells, and the nature of the target cell are all critical variables in determining the characteristics of the cytotoxic event in this system. Spleen cells and bone marrow cells from congenitally athymic nude mice as well as from their heterozygous control littermates were capable of mediating lysis of RBC targets in the presence of either PHA or Con A. Removal of macrophages from these effector populations by adherence columns, density gradient centrifugation, and carrageenan treatment failed to abrogate this cytotoxic capacity. However, purified macrophages themselves also were capable of mediating mitogen induced killing of RBC targets, although the kinetics of this cytotoxicity were substantially different from that induced by lymphocytes. In contrast to these observations, the capacity of mitogen stimulated cells to kill metabolically active complex targets like the P815 mastocytoma or cultured L cells appears to be exclusively a T lymphocyte dependent function. In addition, blastogenic transformation of the effector cells with the T cell mitogens PHA and Con A, but not with the B cell mitogen LPS, leads to enhanced killing of these complex targets. These data suggest that mitogen or lectin induced cellular cytotoxicity can detect at least three different active effector cell types (B cells, T cells, and macrophages) acting via at least four different mechanisms.     

Separation of polypeptides which induce a mitogen response of thymocytes from the culture supernatant of a thymic epithelial cell line

To examine thymic hormonal factors, four polypeptide fractions (estimated molecular weight: I, 10 K; II, 7 K; III, 3 K; IV, 2.5 K) were separated from the culture supernatant of a rat thymic epithelial cell line by high-pressure liquid chromatography (HPLC) with a gel-filtration column. The effects of the fractions on response to mitogens of three small-lymphocyte subsets were studied. All fractions enhanced response to concanavalin A (Con A) of the lighter subset containing mainly immature thymocytes, but only fractions II and IV increased response to phytohemagglutinin (PHA) of the heavier subset containing relatively mature thymocytes. When fraction IV was subfractionated by reversed-phase HPLC, the polypeptides that enhanced response to Con A and PHA were separated into hydrophobic and hydrophilic subfractions, respectively. Fraction I was subfractionated by a similar method, and the inducing activity of Con A response was found in a relatively hydrophobic subfraction. These data suggested that the cell line secretes several kinds of bioactive polypeptides that affect the thymocytes at different stage of maturation.     

The effects of four mycotoxins on the mitogen stimulated proliferation of bovine peripheral blood mononuclear cells in vitro

The effects of four mycotoxins, T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA) and fumonisin B1 (FB1) on the response of bovine peripheral blood mononuclear cells (PBM) in vitro to the mitogens concanavalin A (Con A), phytohaemagglutinin A (PHA) and pokeweed mitogen (PWM) were assayed after 4 days' incubation using 3H-thymidine uptake and the MTT bioassay. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by 3H-thymidine incorporation was for T-2 toxin 0.30, 0.40 and 0.18 ng ml-1; for DON 0.07, 0.09 and 0.04 μg ml-1; for OTA 0.10, 0.20 and 0.15 μg ml- 1, and for FB1 35, 18 and 11 μg ml-1M by 50% for Con A, PHA and PWM as measured by the MTT bioassay were for T-2 toxin 2.0, 2.0 and 1.0 mg ml-1; for DON 0.70, 0.50 and 0.50 μg ml-1; OTA 1.5, 1,5 and 1.5 μg ml- 1; and FB1 >50, >50 and 20 μg ml-1 respectively. Further cytotoxicity assays including the LDH bioassay and Trypan blue exclusion were performed only on Con A-stimulated PBM cells after 72 h incubation. With the LDH-bioassay the 50% inhibition levels were T-2 toxin 0.3 ng ml-1, DON 0.4 μg ml- 1, OTA 1.4 μg ml-1 and FB1 3.5 μg ml-1; for Trypan blue uptake the 50% inhibition levels were T-2 toxin 5 ng ml-1, DON 2.3 μg ml-1 and OTA 4 μg ml-1 respectively. This revised version was published online in June 2006 with corrections to the Cover Date.