The present report shows that thymocyte death, induced by glucocorticoids, may be modulated in vivo by ouabain. Young, ten days old, mice injected with 140 mg/kg sodium succcinate of hydrocortisone (HC) intraperitonially (i.p.) displayed, 24 h after the injection, a decrease in thymus size and cellular content, an effect that was magnified when ouabain (OUA) 0.56 mg/kg, i.p. was given 1 h prior to the HC injection. Ouabain per se was not capable of producing these changes. Both HC and the combination OUA plus HC induced the death of immature double positive lymphocytes (CD4 + CD8 + ) whereas CD69 + cells survived both treatments. An increase in annexin positive cells and a decrease in mitochondrial membrane potential, assessed by cytofluorimetry, using the fluorescent dye DiOC 6 , was observed in thymocytes from HC treated animals indicating apoptosis of these cells. Furthermore, a synergistic effect between OUA and HC was also observed using this parameter. The synergy observed in the thymus of animals treated with glucocorticoids and OUA might occur under stress, when both hormones are released, or in situations when ouabain is administered exogenously in a moment of the circadian cycle when glucocorticoid levels are elevated. However the impact of this effect on the immune response is still unknown.

Initial ligand selection and the intermolecular spatial arrangement ofglycan-lectin complexes are assumed to be essential to induce formationof stable cell aggregates by a lectin. To distinguish effects of thesetwo processes, the tetrameric mistletoe lectin and its isolated B-chainwere used. A reduced impact of multivalency for Ehrlich ascites tumorcells in contrast to rat thymocytes was revealed. Signaling is thusinitiated in a cell-type-dependent manner. Using selective metabolicinhibitors to reduce signal transfer for aggregate stability, decreasein cellular SH-group level was shown to be a common effect accompanyingsuppression of lectin-dependent aggregate stability. The resultsunderscore an intrinsic variability in the relative importance oflectin-dependent glycan aggregation on the cell surface for triggeringpost-binding lectin effects.

In the present work we examined whether the interaction between albumin molecules and thymocytes involves albumin-binding proteins (ABP). Two plasmalemma-rich fractions obtained by differential centrifugation from rat thymus lymphocytes were characterized biochemically and morphologically. These fractions were examined by ligand-blotting and ligand affinity chromatography techniques. Plasmalemma proteins separated by SDS-PAGE were electrotransferred onto nitrocellulose membranes and incubated with 125 I-albumin, in the presence or absence of excess native albumin. The autoradiogram revealed specific binding to two sets of polypeptides of 16–18 and 29–31 kDa, which could be blocked by native albumin. To elucidate whether albumin-binding proteins are exposed on the cell surface, intact lymphocytes were surface radioiodinated and membrane fractions prepared from them were subjected to affinity chromatography on albumin-agarose beads. The proteins thus purified had, like ABP, M r of 16 and 31. These data indicate that ABP (i) are components of thymocyte plasma membrane, (ii) have apparent molecular mass of 16–18 and 29–31 kDa, and (iii) are exposed on the outer membrane surface.

Balb/c mice were infected intraperitoneally with protoscoleces of Echinococcus granulosus . After 15 months of infection, and by means of flow cytometry, the expression of T-cell markers CD3, CD4, and CDS on T cells from peripheral blood, spleen, and thymus was analyzed and compared with that of age-matched controls. Infected mice had higher percentages of CD3+, and CD4+ cells in peripheral blood, and higher percentages of CD8+ cells in the spleen, when compared with control mice. CD4+ and CD8+ cells in peripheral blood and CD8+ cells in thymus also showed higher percentages of expression of interleukin-2 receptor. The results infer a role for interleukin-2 in experimental secondary echinococcosis.

The short-term effects of high concentrations of Methylprednisolone (MP) on the energy metabolism of quiescent and Concanavalin A-stimulated rat thymocytes were investigated in vitro. Concanavalin A (ConA) stimulated the respiration rate of quiescent thymocytes by 35%. Addition of more than 0.15 mg MP/10 7 cells to ConA-stimulated cells reversed this respiratory stimulation; in addition, higher concentrations of MP caused a similar progressive decrease in the rate of respiration of both quiescent and ConA-stimulated cells. Similarly, the stimulation of respiration by ConA was greatly reduced in MP-treated cells. MP addition lowered cytoplasmic [Ca 2+ ] and, at high concentrations, abolished the ability of ConA to increase [Ca 2+ ]. Thus MP both reverses and prevents the immediate stimulation of thymocytes by ConA. In quiescent thymocytes, MP strongly inhibited that part of the oxygen consumption used to drive the cycle of Na + influx across the plasma membrane and Na + efflux on the Na + K + -ATPase, but did not inhibit oxygen consumption used to drive protein synthesis. In ConA-stimulated thymocytes MP had the same effects and also strongly inhibited oxygen consumption dependent on the cycle of Ca 2+ influx across the plasma membrane and Ca 2+ efflux on the Ca 2+ -ATPase, but had little effect on oxygen consumption used to drive RNA and DNA synthesis. These results show that MP prevents cation cycling in thymocytes (either by preventing cation influx or by inhibiting cation pumps) and prevents mitogenic stimulation of the cells. The high MP concentration required and the speed of onset of the effect (lless than 30s) provide strong evidence that these effects of MP are not mediated by glucocorticoid receptors and subsequent activation of gene expression. They may be caused by direct effects of MP on the properties of the plasma membrane. These effects are considered to be, at least partially, responsible for the beneficial results that currently have been obtained using MP megadoses in various clinical situations.

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na + K + -ATPase, Ca 2+ -ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na + K + -ATPase with about 20% each of total oxygen consumption, while Ca 2+ -ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na + K + -ATPase, Ca 2+ -ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na + K + -ATPase with about 20% each of total oxygen consumption, while Ca 2+ -ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.

Thymocyte growth peptide (TGP) promotes DNA synthesis of immature thymocytes. TGP has been purified from sheep, human and calf thymus and recently characterized as an N-terminally blocked nonapeptide. Evidence is presented here that the blocking moiety consists of a formylpteroyl group bound to the N-terminal glutamyl residue of the nonapeptide. The pterin part of the TGP molecule has a ribityl substituent in analogy with riboflavin, which explains the pronounced hydrophilic property of TGP in contrast to unsubstituted and unconjugated folates. The compound can be classified as a ribofolate peptide, a novel class of growth factor. Zn 2+ counteracts degradation of the molecule and is required for full biological activity; mass spectrometric data confirm that native TGP contains zinc.

Gangliosides are normal constituents of the plasma membrane. Exogenous gangliosides can be incorporated into the membrane and extensive research in nervous tissue has demonstrated a beneficial effect of gangliosides on the functional recovery of lesioned neurons and protection against neurotoxins. This paper shows that the effect of gangliosides is not restricted to neurons. The monosialoganglioside GM 1 efficiently increases the survival of thymocytes and protects them against both the lytic effect of the glucocorticoid prednisolone and the effect of a thymocytotoxic serum. The protective effect of GM 1 was achieved both in vitro and in vivo .