Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Studies on the synthetic capacity and antigenic expression of glutaraldehyde-fixed target cells.

Mastocytoma cells (P815 of the DBA/2 strain) treated with increasing concentrations of glutaraldehyde were concurrently evaluated for their ability to incorporate exogeneous uridine, thymidine, and amino acids. The antigenic expression and membrane integrity of these treated cells were assayed by measuring their susceptibility to lysis by antibody and complement and by T-effector cells. The concentrations of glutarladehyde required to effect target cell antigen display were greater than those required to inhibit totally the cell's protein and nucleic acid synthetic processes. Thus, P815 cells treated with 0.15% glutaraldehyde for 10 sec were unable to incorporate either amino acids or nucleotides into macromolecules, but were readily lysed by effector T cell populations, and by alloantibody in the presence of complement. Target cells treated with glutaraldehyde concentrations in excess of 0.25% for 10 sec were resistant to both forms of immune lysis. In keeping with these observations, monolayers of P815 cells treated for 10 sec with 0.15% glutaraldehyde, were capable of specifically depleting T-effector cells from a cytolytically-active spleen cell population. After treatment with higher concentrations of glutaraldehyde (0.3%), however, the monolayers lost their capacity to adsorb effector cells. Although P815 cells treated with glutaraldehyde continued to exhibit H-2d alloantigen, neither these cells nor glutaraldehyde-treated DBA/2 spleen cells induced significant blastogenesis or stimulated the production of cytolytically active effector cells in mixed leukocyte cultures.     

Structure-activity relation for synthetic phenoxazone drugs: evidence for a direct correlation between DNA and pro-apoptotic activity.

The structure-activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. The thermodynamic parameters of drug-DNA complexation were determined by differential scanning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity. Minor modifications in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked structure-activity relation.     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Stimulation of the in vivo dinitrophenyl antibody response to the DNP conjugate of L-glutamic acid60-L-alanine30-L-Tyrosine10 (GAT) polymer by a synthetic adjuvant, muramyl dipeptide (MDP): target cells for adjuvant activity and isotypic pattern of MDP-stimulated response

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.     

Biological activity of synthetic subunits of streptococcus peptidoglycan. III. Relationship of subunit and analogue structure to adjuvant activity in cell-mediated immunity

Each of a series of synthetic peptidoglycan subunits and subunit analogues was injected in combination with streptococcus type M24 antigen extract. The substances tested were: (8a) N-acetylmuramyldipeptide (MDP) and the following derivatives thereof: MDP modified in positions C3 and C4, or with L-alanine substituted by L-2-aminobutyric acid or with the peptide chain prolonged (by three lysines or a polylysine); (b) some synthetically prepared peptides: a hexapeptide, a tridecapeptide and an octadecapeptide. Configurations in positions C3 and C4 were found essential for the adjuvant effect. Adjuvant activity, though somewhat lower than in MDP, was pronounced in the analogue containing the L-2-aminobutyryl residue. Surprisingly, potent adjuvant effect was displayed by the hexapeptide; prolongation of the peptidic chain was not effective. The use of a polymeric carrier for MDP increased the adjuvant effect. Contrary to expectation, streptococcal antigens used with immunoadjuvant materials showed that induced delayed hypersensitivity was type related.     

Calcium channels: evidence for oligomeric nature by target size analysis.

Radiation inactivation was employed to measure the molecular size of calcium channels in guinea-pig skeletal muscle membranes, labelled by the potent 1,4-dihydropyridine calcium antagonist [3H]nimodipine. The molecular size was decreased when the membranes were preincubated and assayed with d-cis-diltiazem, a calcium channel blocker, which is structurally unrelated to the 1,4-dihydropyridines. d-cis-Diltiazem, which is a positive heterotropic regulator of 1,4-dihydropyridine calcium channel binding in vitro, reduced the molecular size from 178 000 to 111 500. 1-cis-Diltiazem, the diastereoisomer, which is devoid of calcium antagonistic action, did not decrease the molecular size of the 1,4-dihydropyridine binding site. Neither diastereoisomer affected the molecular size of the membrane-bound acetyl-cholinesterase, indicating that a stereospecific interaction with the calcium channel structure is the basis for these observations. It is concluded that this decrease in size is indicative of the oligomeric nature of the calcium channel and that calcium channel blockers, acting via different, but interacting drug receptor sites, induce different conformations of the channel structure, resulting in altered conductivity for ions.     

Farmer's peptidomimetic approach: A target for synthetic combinatorial libraries

Summary Two combinatorial libraries of 1296 compounds each were synthesized from two sets of carboxylic acid building blocks and two diamino acid scaffolds. The library was designed to produce low-molecular-weight compounds in a soluble form, to be assayed as potential ligands for peptidergic receptors.     

The cellular basis of adjuvant arthritis: II. Characterization of the cells mediating passive transfer

It has previously been reported that lymph node or spleen cells from rats with adjuvantinduced arthritis can transfer the disease to normal recipients after being cultured with concanavalin A (Con A). In this report, it is shown that a subpopulation of cells that (1) lack surface Ig and the antigen reactive with the monoclonal antibody OX8, (2) are largely nonadherent and esterase negative, and (3) are predominantly marked by the monoclonal antibody W3/25 can transfer arthritis after stimulation with Con A. Adjuvant-sensitized lymph node or spleen cells stimulated with Con A but not PHA transfer arthritis, and this difference correlates with relatively higher levels of interleukin 2 secretion by Con A-stimulated cells. A synthetic adjuvant, CP-20961, a substituted propanediamine, induces arthritis that is passively transferable under the same conditions as arthritis induced by classical mycobacterium-containing adjuvant. The data support the hypothesis that adjuvant inoculation in the rat results in the induction of a unique subpopulation of T cells that initiate the inflammatory joint disease.     

Melanogenesis in the pigment cells of Rana esculenta L. liver: evidence for tyrosinase-like activity in the melanosome protein fraction

This work demonstrates the presence of a tyrosinase-like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.     

Development of a bioassay from gonadal cellular cultures of the snail Helix aspersa: influence of nerve ganglion extracts on the synthetic activity of the target cells]

To realize bioassays a primary culture method was carried out with dissociated cells from the garden snail gonads. ADN and protein syntheses of gonadal cells were estimated using the liquid scintillation method. The gonadal cells were obtained from either adults of Petits Gris and Gros Gris, or young Gros Gris. The results were remarkably homogeneous. The brain extracts added to the culture medium had an inhibitory dose dependent effect on the synthetic activity of gonadal cells. The effected bioassays permit quantitative estimation on the variations of the brain extracts effect in relation to the physiological states of the snail and on the evolution of target cells' receptors during the growth of Gros Gris.     

The cytotoxic activity of hematoheme: evidence for two different mechanisms

Hematoheme displays a potent cytolytic activity toward erythrocytes either in the presence of hydrogen peroxide and a halide ion (system I) or in the presence of oxygen and a reducing agent (system II). In system I it resembles the cytotoxic activity of various peroxidases, whereas in system II it resembles the destructive activity of bleomycin and a variety of metal complexes. Both types of reactions presumably involve the generation of active oxygen species, which are responsible for the damaging effects. In a first attempt to compare the chemical mechanisms of the two types of reactions we used various traps and scavengers of active oxygen species. Tryptophan as well as tyrosine and uric acid were found to be potent inhibitors of the hematoheme-H2O2-halide reaction but do not significantly inhibit the hematoheme-O2-ascorbate reaction. Pyridine, on the other hand, inhibits the oxygen-mediated reaction, but does not affect the peroxide-halide-mediated activity. The cytolytic activity of photoactivated hematoporphyrin, which involves the generation of singlet oxygen, is activated by pyridine and is strongly inhibited by diphenylisobenzofuran. The latter compound is a weak activator of both hematoheme reactions. We conclude that the two hematoheme reactions proceed by two different mechanisms and probably generate different toxic intermediates. The results further suggest that the toxic intermediate generated by photoactivated hematoporphyrin (singlet oxygen) does not play a dominant role in either of the two hematoheme reactions.     

Oculomotor evidence for top-down control following the initial saccade

The goal of the current study was to investigate how salience-driven and goal-driven processes unfold during visual search over multiple eye movements. Eye movements were recorded while observers searched for a target, which was located on (Experiment 1) or defined as (Experiment 2) a specific orientation singleton. This singleton could either be the most, medium, or least salient element in the display. Results were analyzed as a function of response time separately for initial and second eye movements. Irrespective of the search task, initial saccades elicited shortly after the onset of the search display were primarily salience-driven whereas initial saccades elicited after approximately 250 ms were completely unaffected by salience. Initial saccades were increasingly guided in line with task requirements with increasing response times. Second saccades were completely unaffected by salience and were consistently goal-driven, irrespective of response time. These results suggest that stimulus-salience affects the visual system only briefly after a visual image enters the brain and has no effect thereafter.     

Langerhans cells: target cells in immune complex reactions.

Skin of normal, cobra venom extract-treated, and C₄-deficient guinea pigs was injected with ferritin-antiferritin or with peroxidase-antiperoxidase immune complexes. Skin and draining lymph nodes were studied to compare the phagocytosis of these immune complexes by Langerhans cells (LC) and by macrophages. When complement was present, immune complexes were damaging to LC, and uptake of the immune complexes, although present, was limited. When components of complement were absent or diminished, increased numbers of LC in lymph nodes were seen, but damage to LC was absent or decreased. However no detectable change in the amount of phagocytosis by LC was noted. Since some LC can carry antigen from skin to lymph nodes and may be involved in the presentation of antigen to lymphoid cells in some cell-mediated immune responses, impairment or abolition of LC function by immune complexes could represent a mechanism through which the local presence of antibodies might interfere with the induction and elicitation of cellular immunity by antigen. Moreover, damage to LC and subsequent release of intracellular (lysosomal?) substances may constitute a general mechanism of response in the skin to injury and may be an integral part of inflammatory and allergic skin reactions.     

Immunohistochemical evidence of progestin target cells in the pituitary gland of ovariectomized rat

Progestin (P) target cells were identified in the pituitary gland of gonadectomized female rats which had been primed with estrogen (E). P staining was localized using the immunohistochemical avidin-biotin-peroxidase (ABP) complex method. Dark brown precipitates were primarily found over the cytoplasm of cells in the pars distalis, but not in the pars intermedia nor in the pars nervosa. The majority of P-sensitive cells in the pars distalis were identical with luteotrophs, a few being lactotrophs. These observations suggest a role of P in the regulation of production and secretion of gonadotrophins in the pituitary glands of female rats.     

Growth-stimulating effect of UF-irradiated blood. I. The radiation dose dependence of the initial growth potencies of blood and of the functional state of the target cells

UV irradiation (UVI) of donor blood in the apparatus used in hospitals of the USSR with the therapeutic aim of autotransfusion of UV-irradiated blood (AUVIB), results in an increase of connective tissue cell growth potency: being added into culture media the supernatants of irradiated blood stimulate DNA-synthetic and proliferative activity of cultured human embryonic cells. The high activity of cells persists for about 2 days. The effect is great with low initial levels of cell proliferative activity. In this case the effect is maximum (about 125% of the control). It is suggested that the above effect may be involved in the mechanism of stimulation of regeneration processes in the organism after AUVIB.