In vivo and in vitro effects of a new macrolide antibiotic roxithromycin on rat liver cytochrome P-450: comparison with troleandomycin and erythromycin

The effects of a new macrolide antibiotic (Roxithromycin) and one of its major metabolite (RU 39001) on rat hepatic drug metabolizing enzymes were compared to those of erythromycin, erythralosamine and troleandomycin (TAO) both in vitro and in vivo. In contrast to erythromycin, erythralosamine and TAO, roxithromycin and its metabolite RU 39001 exhibit: (i) a very poor affinity for rat liver cytochrome P-450, (ii) an unability to be metabolized into a stable inhibitory metabolite-cytochrome P-450 complex and (iii) a decreased ability to induce liver cytochrome P-450 PCNE, an isozyme implicated in drug associations involving some macrolide antibiotics.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Immune response in mice exposed to low-dose X-rays

The present paper was conducted to evaluate the immunological effect of low dose gamma-irradiation. The splenocytes of mice (C57BL/6N), 24 hours after the irradiation from 0.087 to 0.87 Gy, were incubated with mitogens of T or B lymphocytes, allo-antigen (splenic cells of BALB/c mice) (MLC) or sheep red blood cells (SRBC) 10 days after immunization with the SRBC in vivo, and then their incorporations of 3H-thymidine were measured. On the other hand, this incorporation in the presence of T-cell growth factor (IL-2) in vitro and a drug of AET in vivo was investigated to examine their radioprotective effects. The dose-response relation, i.e. decrease of 3H-thymidine incorporation in function of increase of the irradiation dose, was demonstrated in these immunological examinations except at the dose of 0.087 Gy. More, their incorporation was remarkably promoted by the administration of the T cell growth factor and the drug, therefore, these substances represent the radioprotective effect.     

A Eukaryotic Molecular Target Candidate of Roxithromycin: Fungal Differentiation as a Sensitive Drug Target Analysis System

Roxithromycin (RXM), active against prokaryotes, has beneficial side effects such as anti-cancer activities on mammalian cells, but the mechanisms underlying these effects remain unclear. We found that RXM inhibited the cellular differentiation of the rice blast fungus Magnaporthe oryzae. Hence, we screened the targets of RXM by the T7 phage display method with fungal genomic DNA, and identified MoCDC27 (M. oryzae Cell Division Cycle 27) as a candidate. We generated mocdc27 knockdown mutants that the appressoria formation was less affected by RXM. A complemented mutant restored sensitivity against RXM to the level of the wild type. These results suggest that MoCDC27 was involved in the inhibition of appressorium formation by RXM, and that the complex of RXM-MoCDC27 affected another molecule involved in appressorium formation. The T7 phage display method with fungal genomic DNA can be a useful tool in the quest for drug target.     

A macrolide antibiotic,roxithromycin, inhibits the growth of human myeloid leukemia HL60 cells by producing multinucleate cells

The antiproliferative effect of roxithromycin (RXM) was studied using human myeloid leukemia HL60 cells. RXM inhibited the growth of HL60 cells in a concentration-dependent manner, and significantly inhibited growth at concentrations above 75 M. This growth inhibition was not associated with specific cell cycle arrest and DNA synthesis was not impaired. In addition, the number of viable cells remained almost unchanged in the presence of 100 M RXM. RXM induced growth inhibition at least partly by the formation of multinucleate cells. Both flowcytometric and morphological examination revealed that more than 40% of the RXM-treated cells were binucleate. These findings demonstrate that RXM is a potent new modulator of cell cycle progression in HL60 cells and suggest that the inhibition of cytokinesis by this drug may provide a new model for studying mitosis.     

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins.

Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.     

Phosphonoacetic acid: inhibition of transformation of human cord blood lymphocytes by Epstein-Barr virus.

Phosphonoacetic acid disodium salt (PAA) inhibited the transformation of human cord blood lymphocytes by Epstein-Barr virus (EBV) at concentrations of 50-100 microgram/ml. At these concentrations, PAA had no effect on the multiplication of EBV transformed human lymphoblastoid cells or on the survival of human cord blood lymphocytes. The transformation of human cord blood lymphocytes by the B95-8 strain of EBV was measured by 3H-thymidine uptake, 5 days or more after infection. The degree of inhibition of transformation was correlated with the relation between the input of EBV and the concentration of PAA in the experiment. PAA inhibited the transformation even when added 24 h after EBV infection, but had no effect when added 48 h after EBV infection. The inhibitory effect of PAA could be overcome by its removal and normal 3H-thymidine uptake was restored even after 6 days of inhibition. The specificity of the inhibitory effect on EBV induced transformation of human cord blood lymphocytes is discussed.     

Rabbit B spleen lymphocytes and T helper cells. I. Responsiveness to mitogens of B cell subpopulations of different sedimentation velocities and subpopulations bearing or lacking Fcgamma receptors.

The response to anti-allotype (anti-Ab4), Nocardia Water Soluble Mitogen (NWSM), pneumococcal polysaccharide type III (SSS III), and human Fc fragments of various purified and unfractionated rabbit spleen cell populations was determined in terms of 3H-thymidine up-take. B cells were isolated either from untreated suspensions of spleen cells or from suspensions from which adherent and phagocytic cells were removed. The purification factor was greater than the enhancement of 3H-thymidine uptake by anti-Ab4, NWSM, and SSS III as compared with the response of unfractionated spleen cells. It thus appears that a helper cell was involved: the mitogen response of purified B cells was enhanced by the addition of T cells. B subpopulations were separated by sedimentation or by rosetting, which allowed us to separate Fcgamma receptor-bearing cells from cells that did not possess this receptor. There were differences between cells responding to B mitogens not only in sedimentation velocity but also in the absolute number of cells. B cells bearing the Fcgamma receptor were less responsive to anti-Ab4 and more responsive to SSS III, NWSM, and human Fc than were B cells lacking the Fcgamma receptor.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro II. The relationship of selective binding to cytolysis

5′-Methylthioadenosine (MTA), a degradation product of S-adenosylmethionine, inhibits DNA and protein synthesis as well as cellular proliferation in human lymphocyte cultures stimulated with mitogens, antigens, or allogeneic cells. MTA (10^?3M) inhibited [³H]Tdy uptake in PHA- or Con A-induced transformation greater than 95%, and inhibited the uptake of both [³H]Tdy and [¹⁴C]Leu to the same degree in lymphocytes stimulated with PPD or allogeneic lymphocytes. MTA inhibition was dose dependent, inhibition being lost when exogenous levels reached approximately 10^?5M. The inhibitory effects of MTA were not produced by cytotoxicity since MTA-inhibited cells washed free of this compound could be stimulated at least as well as uninhibited cells. Understanding the mode of action of MTA and the mechanisms controlling its metabolism may lead to new approaches for regulating cellular proliferation.     

Roxithromycin downmodulates Th2 chemokine production by keratinocytes and chemokine receptor expression on Th2 cells: its dual inhibitory effects on the ligands and the receptors

Roxithromycin (RXM), an anti-bacterial macrolide, has various immunomodulatory activities. To investigate the ability of RXM to downregulate skin-infiltration of T-lymphocytes, we examined the effects of RXM on keratinocyte production of chemokines and T cell expression of chemokine receptors. Normal human and HaCaT keratinocytes were cultured with RXM and stimulants. RXM at 1 or 10 microM significantly suppressed the production/expression of Th2 chemokines MDC and TARC in these keratinocytes, but the production of IP-10 was not affected. The effect of RXM on T-cell expression of the corresponding chemokine receptors was also tested in Th2-rich peripheral blood lymphocytes. The IL-2-enhanced expression level of Th2 chemokine receptor CCR4 was decreased by RXM at 10 microM, whereas the expression of CXCR3 was unchanged. Thus, RXM downmodulates both the production and receptor expression of Th2 but not Th1 chemokines involved in cutaneous immunity, suggesting its beneficial therapeutic effects on Th2-mediated or allergic skin disorders.     

Effect of mitogens on suppression of antibody formation and proliferation in dense cultures

A study was made of the effect of mitogens on general proliferation and primary immune response to sheep red blood cells in density-inhibited cultures of mouse spleen cells. The mitogens applied included fetal calf serum and both B cell- and T cell-specific mitogens (dextran sulfate, LPS and ConA). Experiments with 3H-thymidine incorporation demonstrated that the proliferation was equally enhanced by any mitogen in both optimal and density-inhibited cultures. The mitogens did not remove the density inhibition of antibody formation.     

Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Cross-linking of T cell mitogens: effects on B and T cell proliferation and immunoglobulin synthesis.

The T cell mitogens Pa-2, concanavalin A (con A) and its dimeric derivative succinyl-con A, were each cross-linked with the bifunctional reagent dimethyl suberimidate. Although the dose-response curves of these insoluble aggregated products were markedly changed from those of the soluble mitogens, each aggregate continued to stimulate DNA synthesis by murine thymus and T cells. Both aggregated Pa-2 and aggregated succinyl con A stimulated DNA synthesis by B cells from athymic (Nu/Nu) mice. Aggregated con A did not stimulate these cells and, like soluble con A, depressed the background incorporation of 3H-thymidine. Unlike soluble Pa-2, aggregated Pa-2 also greatly increased Ig production by both the B cell cultures and B + T cell cultures from normal (BALB/c) mice.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Subpopulations of lymphocytes to produce various lymphokines. I. Function of subpopulations separated by velocity sedimentation.

Identification of a subpopulation of lymphocytes producing lymphokines was attempted by fractionating the lymph node cells from guinea pigs immune to DNP-BSA by velocity sedimentation at 1 x G. Each of six fractions obtained by this procedure was cultured with or without the presence of antigen, and the culture supernatants that were separated 24 hr later were assayed for various lymphokine activities. Most of the lymphokines, including migration inhibition factor, chemotactic factor for neutrophils, mitogenic factor, and lymphotoxin were generated by the first two fractions of lymphocytes, which represented the largest, most rapidly sedimenting cells. Although th procedure of cell separation does not depend on cell surface properties, the larger cells contained more cells with T cell surface markers and the smalller contained more cells with B cell surface markers. Proliferative response of those lymphocytes measured by 3H-thymidine uptake, however, has shown that the largest two subpopulations responded poorly either to specific antigens or to mitogens (PHA and LPS), and rather that the medium size cells responded most strongly to the both stimulants. These results indicated that the production of some lymphokines confined to certain subpopulations of lymphocytes which are large in size. Further, these cells are readily separable from the medium sized cells that respond strongly to antigenic and mitogenic stimuli with mitogenic responses.     

Active substance of Histoplasma capsulatum that inhibits blastogenic response of lymphocytes

A substance inhibiting blast transformation of murine spleen lymphocytes stimulated with various mitogens, such as LPS, PHA, and PWM, was obtained from yeast-form cells of Histoplasma capsulatum. This active substance was partially purified from the cell-free extract by DEAE-Sepharose CL-6B column chromatography. As a result of this partial purification, the inhibitory activity was 1.26 micrograms/ml in terms of ID50. Materials from H. capsulatum also inhibited blast transformation of murine spleen lymphocytes stimulated with the antigen PPD as well as mitogens LPS, PHA, and PWM. However, the con A-induced proliferative response was only slightly affected. A similar result was observed for the MLR. These inhibitory activities were abolished by heating at 70 C for 30 min. These results suggest that the heat-labile active substance produced by H. capsulatum might directly affect the lymphocytes, leading to inhibition of their blast transformation.     

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.     

Sodium orthovanadate stimulation of DNA synthesis in Nakano mouse lens epithelial cells in serum-free medium

Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H-thymidine at an accelerated rate; the greatest response occurred at 20 microM vanadate, whereas by 2 microM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 microM had lysed by the end of the 40-hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H-thymidine at a vanadate concentration of 200 microM to 500 microM. Half-maximum incorporation of 3H-thymidine (after a 40-hour incubation) was induced by a 2-hour incubation with 20 microM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H-thymidine incorporation, 20 ng/ml insulin in combination with 20 microM vanadate resulted in a significant increase in 3H-thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S-phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S-phase DNA synthesis demonstrated that vanadate was not causing increased 3H-thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H-thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H-thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.