Supraoptimal Doses Research Articles

LHRH analogues can be luteotrophic.

Seven normally menstruating women were treated with consecutive daily supraoptimal doses of the LHRH analogue Hoe 766 initiated in the mid/late luteal phase. This treatment caused immediate increases in circulating levels of LH, FSH, oestradiol (E2) and progesterone. After a few days, the pituitary became unresponsive to Hoe 766 and LH and FSH levels returned to the normal luteal phase ranges while progesterone and E2 levels did not decline to their respective pre-treatment levels until at least 24 h later. As a result of this treatment, the onset of the next menses was delayed in five of the seven women, indicating luteotrophism. These data show that the corpus luteum was responsive to increased LH/FSH during Hoe 766 therapy. Therefore, reported luteolytic effects of short-term treatment with LHRH and/or its analogues were probably not caused by direct inhibitory ovarian action but by reduction of ensuing pituitary gonadotrophin levels.

The effect of extracellular calcium deprivation on amylase secretion and 45Ca efflux from rat pancreas.

1. The role of extracellular Ca(2+) in pancreatic enzyme secretion and (45)Ca efflux evoked by acetylcholine (ACh) and caerulein has been assessed in the incubated, uncinate pancreas of young rats.2. In a medium containing 2.5 x 10(-3)m-Ca(2+), the maximal rates of amylase secretion evoked by optimal doses of each secretagogue were similar. However, the time courses of amylase release during prolonged stimulation of the gland were different.3. The time course of amylase secretion in response to an optimal dose of ACh (10(-5)m) was characterized by an initial rapid increase followed by a slow sustained rise. For caerulein (10(-8)m), an initial rapid rise was followed either by a plateau or slight decline in the rate of amylase secretion.4. Both secretagogues produced similar increases in the rate coefficient of (45)Ca efflux from the gland.5. With supra-optimal doses of the secretagogues, amylase secretion, but not the rate coefficient of (45)Ca efflux, was depressed.6. Reducing the extracellular Ca(2+) concentration did not have a marked effect on basal amylase secretion but inhibited the action of both secretagogues. When the Ca(2+) concentration was 10(-6)m or lower, these inhibitory effects were irreversible. Amylase secretion stimulated by ACh was more sensitive to extracellular Ca(2+) deprivation than that stimulated by caerulein, the concentration required for half-maximal secretion being about 9-fold greater for ACh.7. Decreasing the extracellular Ca(2+) concentration increased both the basal and stimulated rate coefficients of (45)Ca efflux.8. Our results support the hypothesis that pancreatic enzyme secretagogues act by releasing bound Ca(2+) from sites within the acinar cell. Furthermore, they suggest that the site utilized by ACh is more amenable to depletion, via changes in extracellular Ca(2+) concentration, than that employed by caerulein.

Modulatory effects of splenic accessory cells on in vivo antibody response.

We have previously reported that the transfer of 2 x 106 radioresistant (r.r.) spleen cells into normal syngeneic recipients enhanced antibody response to SRBC in rats (1). Similarly, an increased PFC response to 5 x 10 SRBC was induced in BALB/c mice by the i.p. injection of 1–5 x 106 r.r. syngeneic spleen cells, at the time of immunization (2). Elimination of adherent or phagocytic cells abolished the enhancing capacity of r.r. cells suggesting that cells responsible for the observed effect were macrophages. One hour preincubation in medium containing 0.4 mg/ml, kappa-carrageenan potentiated rather than inhibited the enhancing effect of r.r. spleen cells. The transfer of r.r. cells significantly increased the number of PFC in the spleens of mice immunized with suboptimal dose (5 x 105 SRBC) but did not abrogate the suppressive effect of the supraoptimal dose of antigen (5 x 109 SRBC). In order to test whether the potentiating effect of accessory cells is dose-dependent, graded numbers of r.r. spleen cells were injected into mice immunized with 5 x 107SRBC.

Modulation of Suppressor Mechanism in Allergic Contact Dermatitis

We have previously shown that Corynebacterium parvum treatment reduced tolerance induced by intravenous injection of 2,4-dinitrobenzene sulfonic acid (DNBSO3) or by sensitizing BALB/c mice with a supraoptimal dose of 2,4-dinitrofluorobenzene (DNFB). Further analysis by transfer experiments revealed that the generation and/or functional expression of suppressor T-lymphocytes (Ts-cells) was inhibited in both tolerance models. Inhibition of Ts-cells was not entirely correlated with suppression of mitogen induced lymphocyte proliferation mediated by C. parvum activated macrophages. In this investigation we studied the effect of serum obtained at various times after C. parvum injection on tolerance and Ts-cells in DNFB contact allergy. Serum obtained 6 and 24 hr, however, not 72 hr after C.parvum injection inhibited Ts-cells, induced by DNBSO3 and tested by transfer to naive recipients, as efficiently as C. parvum itself. The serum had following characteristics: (1) It inhibited the functional expression of Ts in the recipient animal. (2) It inhibited tolerance induction by an epicutaneous supra-optimal dose of DNFB. (3) It did not inhibit the induction and functional expression of T-effector cells of delayed hypersensitivity (TDH-cells) as shown by transfer experiments. (4) The Ts-inhibitory factor was heat resistant (56 degrees C), not destroyed or lost by dialysis against tris-glycine buffer pH 2 and could not be detected in the serum of NMRI-mice injected 24 hr before with C parvum. (5) The C parvum serum did not significantly increase the spleen weight or suppress mitogen-induced spleen lymphocyte proliferation. The nature of this Ts-cells inhibitory factor is unknown, although the results suggest that cortisone or transfer of bacteria are not responsible. Factors mediating selective inhibition of Ts-cells may be of important regulatory function in delayed type hypersensitivity.

Role of T cells in the mitogen-induced proliferation and polyclonal antibody response of murine B cells.

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in the nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.

Modulation of Suppressor Mechanisms in Allergic Contact Dermatitis: 1. Effect of C. parvum on the Induction Phase of Contact Allergy

The effect of a pretreatment with corynebacterium parvum (C. parvum) on contact allergy in BALB/c mice was studied. Mice sensitized with 50 microliter (supraoptimal dose) 2.4-dinitrofluorobenzene (DNFB, 0.5%) showed a suppressed response as measured by ear swelling after painting the right ear with 0.3% DNFB in comparison to an allergic response obtained with an optimal sensitization dose (15 microliter DNFB 0.5%). By transfer of spleen cells from donors sensitized with a supraoptimal or an optimal dose to recipients either challenged ro sensitized shortly afterwards with DNFB it could be shown that less functionally active immune T-lymphocytes of the delayed hypersensitivity type and significantly more suppressor T-cells were induced in supraoptimally sensitized mice in comparison to the optimally sensitized animals. Intraperitoneal injection of C. parvum (2.8 mg/mouse) one week before sensitization enhanced the contact allergic response in mice sensitized with a supraoptimal dose of DNFB, with little effect on the response in optimally sensitized animals. Further analysis of this enhancement in transfer experiments showed that C, parvum selectively suppressed the generation and/or functional expression of T-suppressor cells and, probably by this mechanism, increased the number of functionally active T-immune lymphocytes. It is proposed that possibly by activation of the immune system C. parvum will suppress suppressor cells in contact allergy and by this mechanism might facilitate sensitization to the contact allergen.

Modulation of concanavalin A-induced lymphocyte stimulation by human low-density lipoproteins.

Spleen lymphocytes and T cells were stimulated by concanavalin A in the presence of various concentrations of human low-density lipoproteins (LDL). The proliferative responses were measured by [14C]thymidine incorporation. Low LDL doses (5-50 microgram/ml) significantly enhanced the stimulation of splenic lymphocytes and T cells. Further, LDL had the capacity to partially relieve the suppression produced by supraoptimal doses of concanavalin A.

Immunosuppressive Lymphocyte Factors: Characterization of the IDS-Producing Cell in the Experimental Model of Antigenic Competition

Inhibitor of DNA synthesis, is a soluble, protein lymphocyte factor which nonspecifically suppresses in vitro lymphocyte responses to antigens or mitogens. It is secreted in large amounts in vivo in some experimentally induced immunological paralysis. Here, we have defined the cell secreting IDS in one experimental model of non-specific immune-suppression, ie., that of antigenic competition. Lymphocytes of rats injected with a large dose of ovalbumin intravenously, show no immunologic response to the same or other antigens or mitogens 24 hr later. At this time, spleen cells of these rats secrete large amounts of the inhibitor into culture supernatants. However spleen cell supernatants of T-depleted rats do not contain the activity. Further, maximal inhibitor concentrations are obtained in the first 2 days of culture when more than 65% of cultured cells are large blasts actively synthesizing protein. As the number of actively metabolizing blast cells decrease in subsequent days of culture inhibitor concentration falls. Finally thymocytes of rats pretreated with hydrocortisone acetate, to deplete thymus cortex cells are unable to secrete inhibitor in culture. These findings reveal that the cells producing inhibitory DNA synthesis in an animal made tolerant with a supra-optimal dose of antigen is an active blast transformed T cell (present in the spleen and thymus). In the thymus the cell making inhibitor appears to reside in the thymus cortex. Previous experiments have confirmed that an identical cell causes nonspecific immune-suppression in vitro. We suggest that this cell produces in vivo tolerance in antigenic competition through the release of inhibitor to DNA synthesis.

Changes in lymphocyte populations in protein—Calorie-deficient mice

Changes in lymphocyte subpopulations induced by postweaning malnutrition were studied in C57BL/6 mice kept on a protein-restricted diet (D), by weekly assessment of the “homing” properties and the response to mitogens of thymus and spleen lymphocytes during the first 2 months of diet. Cell loss in the lymphoid organs during the early phase of protein restriction was mainly due to depletion of nonrecirculating cells. This resulted in relative enrichment of medullary cells in the thymus and T 2 cells in the periphery as shown by the rise in the percentage migration of D lymphocytes to the lymph nodes as well as in their response to optimal doses of PHA and Con A and PHA:Con A response ratio. Reversion of the distribution pattern of D lymphocytes, with depressed homing to the lymph nodes and decrease in the response to mitogens, was observed concomitantly with a second phase of partial recovery in the whole-body weight and cell content of the thymus and spleen. The [ 3H]thymidine uptake by D spleen cells stimulated with supraoptimal doses of mitogen was significantly increased during the whole length of the experiment. The suppression of DNA synthesis induced by high doses of mitogen reappeared after short-term nutritional rehabilitation.

Influence of Sub- and Supra-Optimal Concentrations of Some Growth Regulators On Growth of Guava, Phenol Status, Nitrogen Concentration and Numbers of Meloidogyne Incognita

The effects of several plant growth substances on the growth of guava, root-knot development, phenolic content and nitrogen concentration were studied using sub- and supra-optimal doses applied as foliar sprays. Shoot and root growth were stimulated by sub-optimal doses of growth retardants and promotors but retarded by supra-optimal ones. Sub-optimal doses of CCC and daminozide plant retardants stimulated stem elongation and internode length and increased the numbers of nodes, branches and leaves. Supra-optimal doses of ethephon and GA plant stimulators had the reverse effects. The multiplication of Meloidogyne incognita at sub- or supra-optimal rates of growth retarding or stimulating substances was greater in treated than in untreated plants, but the degree of root-galling was sometimes independent of population density. Effectiveness of plant growth regulators on host growth and associated nematodes seems largely dosage dependent. There was an accumulation of phenols in treated tissues compared with those translocated or synthesized in untreated roots. Some 26.5 times more accumulated phenols were observed after a CCC subdose than in untreated controls. The mono/poly phenol ratio seemed to influence root galling. Nitrogen concentration varied little.

Flowers of Ipomoea tricolor as indicators of ethylene inducing compounds

Summary Ephemeral flowers of Ipomoea tricolor were tested with regard to their suitability for the rapid screening of senescence inducing chemical compounds. The system was standardized by the ethylene releasers 2-CEPA, CGA 17856 and CGA 15281 which accellerate the fading process, manifested by rolling up of the corolla, considerably. ACC, a precursor of ethylene biosynthesis in apple tissue, had an even more pronounced effect on the corolla. IAA, a known ethylene inducer when applied in supraoptimal doses, hastened the fading process noticably. Its conjugates with methionine and aspartic acid did not exceed the effect of IAA. IAA-N e -lys was almost as active as IAA itself, whereas IAA-N α -lys was of no importance in senescence. Rolling up of the corolla is inhibited by cycloheximide. However, AVG (ethyl analog of rhizobitoxin) an inhibitor of ethylene biosynthesis does not have the capability of cancelling the biological action of ACC. The various compounds that cause premature curling reactions in flowers evoke the production of ethylene in flower buds approaching maturity.

Cell interactions that regulate the T-cell dose-response profile to concanavalin A.

We have tested the ability of structures on macrophage (M phi) membranes (M phi-MEM) and on several other types of cells with Fc receptors to affect the DNA synthetic response of concanavalin-A-stimulated T cells. Of the cells tested, only M phi-MEM have the capacity to relieve the suppression produced by supra-optimal doses of the mitogen. The M phi-MEM do not increase Con A responses by altering the stimulatory capacity of the Con A. These observations, analysed together with previous results, which have indicated that live intact M phi are required for the transfer of information between lymphocyte sets and that M phi-MEM preparations can act as competitive antagonists for this function, are interpreted as follows: some supraoptimal doses of Con A activate suppressor cells, which are responsible for limiting the DNA synthetic response to the mitogen; the M phi-MEM abrogate this suppression by absorbing the signal that activates the suppressor cell. Kinetic studies suggest that the M phi-MEM do not affect the activity of already activated suppressor cells. We also found that Con A usually activates two separately responding T-cell populations with different sensitivities to dose and to time of contact with mitogen and that the suppression of both populations can be relieved by M phi-MEM. These results support the notion that the overall immunological circuit is composed of multiple independently regulated mini-circuits, with M phi acting as transmission posts for intra- and perhaps also inter-circuit communication.

Immunosuppressive Activity of Canine Pulmonary Surface Active Material

Abstract The effects of canine surface active material (SAM), a lipoprotein unique to the lung, on the proliferative response of lymphocytes to immune stimulation were investigated. Peripheral blood lymphocytes were cultured in the presence of either endobronchial lavage fluid or purified SAM extracted from such lavage fluids. Cultures were stimulated with mitogens (Con-A, or PWM) or allogeneic cells (MLC), and the proliferative responses were measured by the uptake of tritiated thymidine. Culture of lymphocytes in autologous endobronchial lavage fluid resulted in a suppressed response to all three stimuli: MLC (33% control response), PWM (54% control), Con-A (75% control). Culture of lymphocytes with purified SAM produced a dose-dependent inhibition of the responses to these stimuli (MLC > PWM > Con-A). Fifty percent inhibition of lymphocyte responses to MLC, PWM, or Con-A was produced by 4.2, 7.5, or 12.5 µg protein/ml of purified SAM, respectively. Supraoptimal doses of mitogens failed to alter the observed inhibitory activities. SAM was not cytotoxic for lymphocytes in these experiments. SAM was most inhibitory when added to lymphocytes at the time of mitogen stimulation; progressively less inhibition was produced by SAM added at increasing intervals of time after stimulation. Lymphocytes preincubated with SAM for 24 or 48 hr, washed, and then cultured in fresh medium displayed persistently suppressed responses. These results demonstrate that canine SAM is a potent suppressor of lymphocyte proliferation in vitro. In vivo, SAM is found only in the lung and, during fetal development, is secreted by the lung into amniotic fluid. The presence of SAM in these locations may have important implications for our understanding of pulmonary and maternal-fetal immunology.

Suppression of Humoral and Delayed Hypersensitivity Repsonses by Distinct T Cell Subpopulations

Abstract Immunization of mice with a supraoptimal dose of sheep red blood cells (SRBC) results in splenic T cell populations capable of specifically suppressing recipients' plaque forming cell (PFC) and delayed-type hypersensitivity (DTH) responses to SRBC when tested in an adoptive transfer system. By localization on discontinuous bovine serum albumin (BSA) gradients and relative sensitivity to Cytoxan, two distinct T cell subpopulations suppressing DTH reactivity were identified. One population could not be distinguished from T cells capable of inhibiting direct and indirect PFC responses. However, another population appeared quite distinct and capable of inhibiting DTH, but not PFC responses.

EFFECT OF AZATHIOPRINE AND PREDNISOLONE ON PASSIVE ENHANCEMENT OF RAT RENAL ALLOGRAFTS

Passive enhancement provides only partial suppression of rejection in the (DA X Lewis)F1 to Lewis renal allograft model. Suboptimal (8 mg/kg/day) and supraoptimal (30 mg/kg/day) doses of azathioprine administered with enhancing serum failed to suppress the rejection reaction in enhanced animals. Similarly, suboptimal (4 mg/kg/day) and optimal (16 mg/kg/day) doses of methylprednisolone were ineffective. However, the onset of rejection in enhanced animals was delayed by the use of both azathioprine (30 mg/kg/day) and methylprednisolone (16 mg/kg/day). The survival times of enhanced animals treated with azathioprine were significantly shorter than those of animals treated with enhancing serum alone, suggesting that this agent may prevent the development of autoenhancement. Although suboptimal doses of antilymphocyte serum suppress rejection in this enhancement model, the dose requirements of conventional immunosuppressive agents appear to be maximal rather than minimal.

Comparative study of DNA elongation in mitogen-stimulated mouse thymocytes and splenocytes

Stimulation of mouse lymphocytes (T and B populations) by specific mitogens (ConA and LPS) results in characteristic biochemical and morphological events. In thymocytes stimulated in vitro with optimal doses of ConA, differentiation processes corresponding to blastic transformation seem to be a prerequisite for DNA synthesis and mitosis. In thymus cells proliferating in vivo, DNA synthesis and mitosis occur even in the absence of blastogenesis. However, the patterns of DNA replication do not differ in either case. The replicon size is evaluated to 45S and the mean rate of DNA synthesis to 0.8 μm/min. The kinetics of integration of replicon is not modified. However, this result could not be presumptive of an absence of modification of these parameters when the lectin is used at supraoptimal doses. Nude mice splenocytes (B cell population) show a pattern of replication which differs from the preceding by the presence of 30S DNA intermediate which is never observed in thymocytes. These pieces could correspond to individual replicons of smaller size or else to a growing intermediate within a replicon, synthesized at a reduced rate. Both of these interpretations are discussed.

Immune Suppression with Supraoptimal Doses of Antigen in Contact Sensitivity

Abstract Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.

Restricted avidity of the IgM antibody response to dextran B512 in mice: Studies on inhibition of specific plaque forming cells by oligosaccharides

A plaque-inhibition test was used to estimate the avidity of the IgM antibody produced by BALB/c mice immunized at different times and with different doses of native dextran B512 (α-1–6 glucan) using as inhibitors high and lower mol. wt polysaccharide fractions (mol. wt 2 × 10 6 and 2 × 10 4) and the corresponding oligosaccharide series from isomaltose up to isomaltohexaose. The avidity of anti-α-1–6-dextran IgM antibody produced appears neither to increase sequentially during the course of the immune response, nor to be dose-dependent over a 1000-fold range of immunizing doses. However, immunization with a supraoptimal dose (100 μg) induced preferentially a response corresponding to isomaltotetraose epitope size, whereas 0.1 μg selected for an isomaltohexaose response. The results suggest that a restricted population of lymphoid cells participates in the antibody response to α-1–6-dextran and that this might limit antigen-dose-dependent selection.

Selective and opposing effects of cytochalasin B and other drugs on lymphocyte responses to different doses of mitogens

Drugs with pharmacologic activities on cell membrane components were tested for their effects on responses of murine lymphocytes to mitogens. The effects of the drugs were found to be partly selective and related to the type and dose of the mitogen. Most striking were the effects of cytochalasin B, which inhibited the responses to low doses of concanavalin A or phytohemagglutinin, but potentiated the reactions to the high doses of the lectins. Chloroquine and chlorpromazine, on the other hand, were more inhibitory to cultures containing supraoptimal doses of the lectins. Colchicine inhibited the responses against lipopolysaccharide more than those against the two lectins. The inhibitory effects of colchicine were almost unchanged when the drug was added at intervals up to 24 hr after the onset of culture, whereas the effects of the other tested drugs diminished markedly when added to cultures 6 hr or more after the mitogens. The results are discussed in view of their relationship to the poorly understood mechanisms which regulate the lymphocyte responses in the presence of different doses of mitogens.

Potentiation of the T lymphocyte response to mitogens: V. Inhibition of the response to Concanavalin A by supraoptimal doses or by other lectins and its aversion by LAF

The enhanced thymidine incorporation in murine lymphocytes induced by Concanavalin A (Con A) was markedly inhibited in the presence of other lectins, which are poorly mitogenic (phytohemagglutinin {PHA} or pokeweed mitogen), or non-mitogenic (soybean agglutinin {SBA}). The level of inhibition was found to be inversely proportional to the mitogenic effect of the lectins. Our results did not support the notions that the lectins inhibit the lymphocyte responses by competing with Con A, or by activating suppressor cells. Rather, the data suggest that the lectins cause cytotoxic or cytostatic effects. The effects of the inhibitory lectins were found to resemble those of supraoptimal doses of Con A. In particular, both effects were partly averted by the lymphocyte activating factor (LAF). The mitogenic effect of LAF was not inhibited by the non-mitogenic lectin, SBA, whereas the poor responses to PHA or to moderately supraoptimal doses of Con A were markedly potentiated by this factor. It is thus suggested that LAF activity counteracts the inhibitory processes provoked by the lectins.