The role of lymphoid dendritic cells (DCs) in the development of an allogeneic cytotoxic reaction in vitro was examined. The T+B and T cell subsets originating from the spleens or lymph nodes of normal and Listeria innocua-infected BALB/c mice were used as the effector cells. Their cytotoxicity to 51Cr-labeled C3H fibroblasts was determined after removal of DCs and replacing them again. Moreover, the influence of exogenous mrIL-12 on the potency of DCs in the allogeneic reaction developed in vitro was checked. It was found that the DC-deprived T+B or T subsets of splenocytes, regardless of their origin, exhibited 27-38% lower cytotoxicity than those accompanied by natural DCs. The cytotoxicity of these subsets from normal lymph nodes decreased by 22%, while the activity of bacteria-primed cells dropped by 38%. Replenishing effector cells with isolated DCs restored their cytotoxicity. Pulsation of normal DCs with IL-12 had no effect on the recovery of normal cell cytotoxicity. However, the IL-12-pulsed DCs were able to intensify the cytotoxicity of T+B subsets derived from the spleens or lymph nodes of L. innocua-infected mice. The results suggest that the alloantigen presentation by DCs to cytotoxic lymphocytes also takes place in the reaction developed in vitro, regardless of effector cell origin.
Introduction: The mechanism of the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth inhibition seems to be highly specific and dependent on cell type and anthracycline drug. Resistance and the high cardiotoxicity of anthracyclines have stimulated many studies aimed at identifying critical substituents required for optimal activity. Many authors point to the fact that the double-strand breaks, the consequence of the activity of topoisomerase II poisons, and the inability of cells to repair the DNA lesions are the signal for apoptosis. The aim of this study was to define the influence of 4-demetoxy 2'-halogenated analogs with altered basicity at the 3'-position on topoisomerase II and the relationship of that interaction with apoptosis and the cytotoxicity of these novel anthracyclines. Parental human ME18 melanoma cells and the ME18/R subline, obtained experimentally, resistant to doxorubicin (DOX), exposed to 1.7 and 8.6 muM DOX or its analogs, annamycin and WP903 (both 0.3 and 3.0 ?M) were studied. Materials and Methods: The MTT test was used to assay cytotoxicity. Interaction of the drugs with topoisomerase II and apoptosis were done by Western blot and fluorescence microscopy using Hoechst 33342. Results: The structural changes at positions 4, 2', and 3' can influence topoisomerase II interaction and apoptotic activity, although correlation between these events and cytotoxic consequences has not been proved. Conclusions: The biological response of the cells to the structurally similar anthracyclines may be variable and probably depends on the cell type which seems to be an additional problem in the multifactorial resistance of tumor cells to anthracyclines.
A critical molecular interaction during assembly of the major histocompatibility complex (MHC) class I molecules takes place between the heavy chain and the transporter-associated with antigen-processing (TAP) complex. The recent mapping of regions of the heavy chain involved in the binding to TAP suggests a complex molecular interaction essential for the cell surface expression of the MHC class I. The advances made in understanding the TAP-MHC class I interaction are reviewed and discussed here.
In mice of the Igha immunoglobulin allotypic haplotype we found, the presence of T lymphocytes with an inherent inhibitory activity against the expression of the IgG2ab allotype (IgG2a of the Ighb immunoglobulin allotypic haplotype). This constitutive anti-IgG2ab T lymphocyte activity can be enhanced in vivo by what we called 'sensitization', which usually consists of one or two intravenous injections of B splenocytes from Ighb congenic mice. When injected at birth, the resulting anti-IgG2ab T splenocytes induce, with 100% success, total, specific and chronic (but experimentally reversible) suppression of IgG2ab in Igha/b F1 hybrid mice prepared by mating Igh congenic mice. Even if restricted to IgG2ab expression, this experimental model, which deals with an unambiguous case of T cell-mediated down-regulation of immunoglobulin production, provides a clear and powerful tool to dissect finely the behavior of the partners (T and B lymphocytes) intervening in regulation within the immune system. For example, we observed that CD4 T lymphocytes were necessary to obtain full recruitment of anti-IgG2ab CD8 T lymphocytes during the sensitization, that suppression induction in anti-IgG2ab T splenocytes of newborn recipients required cooperation between CD4 and CD8 T lymphocytes, and that CD8 T lymphocytes were essential for suppression maintenance. We showed that this suppression was not characterized by an accumulation of B lymphocytes containing the allotype they could not secrete or Cgamma2ab mRNA they could not translate. The recipient's immune system was not involved in the suppession maintenance; this was done by donor T lymphocytes, which ensured the chronicity of IgG2ab suppression throughout the recipient's life. We demonstrated that the mechanism of this suppression implied an MHC-restricted presentation by target B lymphocytes of Cgamma2ab peptides to the T cell receptor (TCR) of anti-IgG2ab T lymphocytes. Notwithstanding the requirement of a CD4-CD8 T lymphocyte cooperation during the induction phase, we functionally determined that the suppression induction implicated an MHC class I-, but not class II-restricted interaction. We also demonstrated the existence in vivo of alternative or concomitant use of perforine- and Fas-mediated cytotoxicity pathways in this T cell-induced IgG2ab suppression. Thus this suppression did not imply silencing IgG2ab production, but B lymphocyte destruction by CD8 T lymphocytes. Always using our suppression model, we demonstrated that an agonistic anti-CD40 treatment helps in recruiting CD8 cytotoxic T lymphocytes, involved in immune regulatory functions and that CD40 expression on Ighb B lymphocytes confronted with CD8 T lymphocyte effectors only operating via the Fas pathway was involved in the total suppression of IgG2ab expression. The selection and maintenance of such normal T cell activity against the IgG2ab allotype in mice of different genetic backgrounds remain somewhat enigmatic. Indeed, we did not observe any similar activity against other immunoglobulin allotypes or isotypes. The intestinal flora had no influence on the emergence of this anti-IgG2ab T lymphocyte activity, as it was untouched in germ-free Igha mice when compared with normal Igha mice. More recently, this model offered an opportunity to study problems pertaining to immune tolerance. For instance, we showed that the genetic elements involved in the building of anti-IgG2ab TCR were available in Igha and Ighb mice of different genetic backgrounds, but that somatic constraints, namely the perinatal presence of IgG2ab, effectively prevented their acquisition, while its absence led to their spontaneous emergence. Consequently, we were able to induce anti-IgG2ab T lymphocytes into a tolerance state by injecting Igha mice with soluble IgG2ab during the perinatal period. However, the full T lymphocyte tolerance obtained in this manner was not definitively acquired, as it had reversed spontaneously when investigated 3 to 6 months after the end of tolerogen treatment, even when this treatment had been prolonged from the perinatal period to 9 months of age. The mechanisms (induction and reversion) of this tolerance involves the physical elimination or the irreversible inactivation of the natural anti-IgG2ab T lymphocyte clones and their resurgence, from bone-marrow precursors, as long as the thymus remains operational, but not the establishment of a reversible, functional unresponsiveness (anergy) or an active, cell-mediated inhibition of anti-IgG2ab T clones. We attempted to elucidate, in Ighb mice, whether the natural T lymphocyte unresponsiveness to IgG2ab involved a central tolerance mechanism and to identify the type of tolerogen implicated in this tolerogenesis. The experiments principally showed that this natural T lymphocyte tolerance to IgG2ab was mediated by a thymic mechanism; that the capacity to induce it was gradually acquired by Ighb thymuses and was most probably due to potentially IgG2ab-producing/presenting cells, progressively colonizing the developing thymus; and that a significantly decreased postnatal Cgamma2ab gene transcription correlated with the emergence of anti-IgG2ab T lymphocytes in Igha/b F1 (postnatally deprived of their B lymphocyte compartment), which subjected them to autoimmune IgG2ab-allotype suppression.
Experiments were carried out on the effect of nickel as an inorganic compound (NiSO4?7H2O) and organic Ni(II) complexes (i.e. Ni(II)-Glu and Ni(II)-EDTA) in concentrations of 20, 40 and 85 muM dm-3 on meristematic cells of root tips of Brassica oleracea L. cv. S?awa from Enkhouizen. All three tested chemical forms of nickel had a mitodepressive effect and inhibited root elongation. With respect to the degree of root elongation inhibition and mitodepressive effect, the tested forms of nickel can be put in the following order: Ni(II)-Glu NiSO4?7H2O Ni(II)-EDTA. In all three tested forms, nickel caused disturbances in mitotic divisions, resulting in anaphase bridges and binuclear cells, whose nuclei were joined by a bridge of condensed chromatin or separated. Inorganic nickel and Ni(II)-Glu in higher concentrations damaged nuclei (the amount of condensed chromatin increased), nucleoli (their structure became more condensed and vacuolisation was observed), endoplasmic reticulum (fragmentation, swelling of cisternae) and mitochondria (structure condensation).
The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 M, for different periods of time (from 30? to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 M concentration. This effect became gradually more evident at higher concentrations ( 28% at 30 M, 50% at 100 M, 68% at 300 M). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 M beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations 23% at 3 M, 47% at 10 M, 65% at 30 M, 90% at 100 M, 99% at 300 M). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 M and 10 M, respectively. In time-course experiments, no effect of beauvericin (30 M) on cell viability could be seen after exposure for periods of time as long as 30?, 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 M. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.
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