In vitro and in vivo murine metabolism of spirogermanium.

We report the identification of spirogermanium (SG) metabolites derived from incubation of the drug with a mouse liver microsomal preparation as well as those obtained from the urine of mice injected with the drug. GC/MS data using electron impact and chemical ionization indicate that the major metabolic products appearing in the urine of mice are hydroxylated metabolites resulting from oxidation of the ethyl substituents on germanium. Thermospray (TSP) LC/MS data suggest that these hydroxy metabolites are further oxidized to an acid and a deethylated metabolite that has undergone hydroxylation of the germanium atom. In a separate experiment, human urine from a subject undergoing therapy with SG was subjected to TSP-LC/MS analysis. The SG metabolite pattern observed in the urine from human was similar to that observed in the mouse urine. These results suggest that the metabolic fate of SG in human is qualitatively similar to that found in the mouse.     

In vitro and in vivo antitumor activity of YoshixolTR against murine L1210 leukemic cells.

In this report, antiproliferative effects of YoshixolTR in vitro and in vivo were investigated in murine L1210 cells. A proliferation of L1210 cells in vitro was inhibited by YoshixolTR in a dose- and time-dependent manner. This inhibition showed an arrest at the G0/G1 stage of the cell cycle, followed by a flow cytometric measurement. YoshixolTR induced apoptosis-like cell death identified by histological observations (scanning electron and transmission electron microscopy), DNA fragmentation, and a smaller increase in lactate dehydrogenase (LDH). In the in vivo experiments, YoshixolTR (5 microl/kg of body weight, on days 1, 3, and 5) was injected intraperitoneally in mice inoculated with L1210 cells. No marked prolongation of survival occurred between the control group and treated group. However, a survival curve in the treated group showed a shift toward a possible longer survival time. Additionally, on the basis of apoptosis-like cell death due to YoshixolTR as indicated above, a possibility of immunotherapy as a tumor vaccine has been examined. A vaccination of rabbit anti-serum, which consisted of components from the L1210 cells killed by YoshixolTR, produced a dramatic improvement of viability in the leukemic mice. In conclusion, YoshixolTR has an anti-leukemic potency with a new biological mechanism and an inductive potency of super-antigens as immunotherapeutic agents against malignant tumors.     

Distinguishing features of cytotoxic and pharmacological effects of selenite in murine mammary epithelial cells in vitro

Previous studies have demonstrated that selenite exerts a reversible and non-lethal inhibition of cell growth which could be correlated with a 58 K selenoprotein. Prolonged exposure to 5 microM selenite is cytotoxic. This cytotoxic effect is heralded by the cells floating off the dish. These floating cells contained over a log more selenite than did the attached cells. The floating cells also had a higher total amount per cell of covalently incorporated selenite in the form of volatile selenides. No specific selenoprotein could be correlated with the cytotoxic effect of selenite. However, a greater aggregation of selenoproteins was observed in dead vs. viable cells. This change probably was a late manifestation of cell death, whereas the increased amount of volatile selenides marked the early stages of acute selenosis and cell death. The results suggest that the cellular response to selenite exposure contains two phases: an early reversible inhibition of cell growth, and a late irreversible cytotoxic effect. The former is characterized by an increase in a 58 K selenoprotein, whereas the latter by an increase in volatile selenides. The results suggest that experiments evaluating specific effects of selenite exposure on biochemical function need to distinguish between the two phases of cell response.     

The effects of immunosuppressive pharmacological agents on the induction of cytotoxic and suppressor T lymphocytes in vitro

The immune system is regulated by the interactions among several distinct functional subsets of T cells. The action of several commonly used immunosuppressive drugs on the activation of cytotoxic T lymphocytes (CTL) and suppressor T lymphocytes (Ts) in the primary mixed lymphocyte reaction (MLR) was investigated. Cyclosporin A, hydrocortisone, and azathioprine were all found to inhibit both CTL and Ts activation when present at pharmacological doses in culture. When these drug-inhibited cultures were reconstituted with interleukin-2, however, clear differences between the effects of these drugs was observed. Cyclosporin A and hydrocortisone allowed the selective activation of Ts in the presence of interleukin-2, while azathioprine inhibition was not reversed by interleukin-2. Thus, CTL precursors appear to be directly inhibited by all of these drugs, but Ts precursors apparently are not inhibited by cyclosporin A or hydrocortisone provided interleukin-2 is present. These findings are discussed in terms of the activation requirements of CTL vs. Ts and the implications of the selective activation of alloantigen-specific Ts for prevention of allograft rejection.     

Strain-dependent in vitro and in vivo effects of oleic acid anilides on splenocytes and T cells in a murine model of the toxic oil syndrome.

The toxic oil syndrome is an exogenously-induced autoimmune disease in humans, which is believed to be due to the accidental ingestion of oleic acid anilides. In a previously established murine model anilides-treated A/J mice developed a wasting disease after 1 week. Anilides-treated B10.S mice showed after 6 weeks a hyperimmunglobulinemia with autoantibody production, but no clinical symptoms. We now compared in vitro the effects of anilides on splenocytes and T cells in A/J and B10.S mice. Splenocyte proliferation was similar in both strains. After in vivo treatment of mice with anilides and in vitro restimulation, splenocytes of sick A/J mice showed a significant increase in splenocyte proliferation. Splenocytes from B10.S mice, however, had a suppressed baseline response and did not proliferate on restimulation. Adherent cells were necessary to induce proliferation in A/J mice-derived T cells. Apoptosis in splenocytes was significantly elevated in anilides-treated A/J and in B10.S mice as compared to saline-treated controls. These data show that anilides are able to affect the immune system in a strain-dependent way and may therefore take part in inducing the disease seen in humans and mice.     

The cytolytic and cytotoxic activities of palytoxin

Palytoxin (PlTX) is one of the largest compound present in nature and, with its strong ability to modify the normal function of different biological systems, is also classified as one of the most potent biotoxins. Many alterations are triggered by PlTX, directly or indirectly related to its interaction with Na⁺,K⁺-ATPase and the consequent conversion of this ion pump into a non-specific cation channel. The resulting perturbation of Na⁺, K⁺, Ca²⁺ and H⁺ ion fluxes is the driving force of PlTX-induced cytotoxic events, culminating with system disruption and, finally, cell death. The modifications in the distribution of these ions across the plasma membrane play key roles in the promotion of the PlTX-induced cytolytic and cytotoxic responses. In this scenario, PlTX-specific cytolysis can be part, but might not necessarily represent a unique aspect of the cytotoxic effects of the toxin. Owing to the complex array of responses, some of them being cell-type-specific and/or affected by experimental conditions, the distinction between cytolytic and cytotoxic events becomes ill-defined, but the two responses show distinct features, whose further characterization could contribute to a better understanding of the molecular mechanism of cellular effects induced by PlTX.     

Soluble components of Utah Valley particulate pollution alter alveolar macrophage function in vivo and in vitro

Water-soluble extracts of Utah Valley dust (UVD) have been found to cause inflammatory injury of the lung in both humans and rodents. The degree of lung damage found correlated with the metal content in the extracts. In the present study, extracts of a set of UVD PM(10) filters collected over a 3-yr span, varying in total metal content with yr 1 = yr 3 > yr 2, were used to assess effects on human alveolar macrophage (AM) function. The phagocytic activity and oxidative response of AM was investigated 24 h after segmental instillation of UVD, or after overnight in vitro culture of the extracts with AM. Using flow cytometry analysis, AM phagocytosis of fluorescently (FITC)-labeled Saccharomyces cerevisiae was inhibited following instillation of UVD1 (61%) but not by yr 2 and 3. Neither baseline oxidant activity nor phorbol ester-induced oxidant generation was affected by the dust extracts in vivo. Overnight culture of AM with UVD1 resulted in a significant decrease in the percentage of AM phagocytizing particles (30%), while no significant effect on this function was found with the other two extracts. Furthermore, only UVD1 caused an immediate oxidative response in AM, although both UVD1 and UVD3 inhibited oxidant activity in AM when the cells were incubated with the extracts overnight. The detrimental effects on AM host defenses could be due to apoptosis, which was evident in cells exposed to the UVD1 and to a much lesser extent with AM exposed to yr 2 and 3. The component(s) responsible for the toxic effects on AM in vitro were removed by pretreatment of the UVD extracts with a polycation chelating resin, chelex-100. However, since yr 1 and 3 are similar in their soluble metal content, but differ in their effects on AM phagocytosis, it is possible that the metals may not be the culprit in effects of particulate matter on AM host defense.     

Immunomodulating properties of prodigiosin 25-C, an antibiotic which preferentially suppresses induction of cytotoxic T cells.

An antibiotic, prodigiosin 25-C, preferentially suppresses cytotoxic T lymphocytes (CTL) without affecting antibody production. Here, we investigated the effect of prodigiosin 25-C on delayed-type hypersensitivity (DTH), graft versus host reaction (GvHR) and allogeneic skin graft rejection. DTH reactions were markedly inhibited by ip treatment of the mice with prodigiosin 25-C. Cell transfer experiments indicated that prodigiosin 25-C exerted its suppressive effect on the late efferent phase rather than on the induction phase of DTH. Prodigiosin 25-C suppressed induction of anti-host CTL when GvHR was induced by iv inoculating splenocytes of parental C57BL/6 mice to adult unirradiated BDF1 mice. It had little effect on GvHR-induced splenomegaly observed 2 weeks after the inoculation, but significantly delayed the subsidence of splenomegaly as revealed 8 weeks later, suggesting that suppression of CTL converts immunosuppressive GvHR to immunostimulative one as reported by G. M. Shearer. However, reduction of interleukin-2 (IL-2) production and mitogen responses induced by GvHR were not rescued by prodigiosin 25-C treatment. Prodigiosin 25-C moderately prolonged survival of major histocompatibility (MHC)-mismatched skin grafts. Since the mode of action of prodigiosin 25-C is distinct from those of cyclosporin A and FK506, these results demonstrate potential usefulness of the antibiotic for a supplementary immunosuppressant.     

Stephanthraniline A inhibits the proliferation and activation of T cells in vitro and in vivo

Stephanthraniline A (STA) isolated from the stems of Stephanotis mucronata (Blanco) Merr. was evaluated for their suppression on T cells' immune responses in vitro and in vivo. In vivo, oral administration of STA significantly inhibited T cell-mediated delayed-type hypersensitivity (DTH) response. In vitro, STA has inhibitory effects on T cell proliferation induced by CD3/CD28 cross-linking or Con A; additionally, CD4(+) T cells are more sensitive to this inhibition than CD8(+) T cells. STA also suppressed the production of cytokines (IL-2, IFN-γ, IL-4 and IL-17) and mRNA expression of the genes associated with T cell activation, proliferation and differentiation. Our data indicate that STA inhibits the proliferation of T cells by inducing cell cycle arrest but not inducing apoptosis. The inhibitory mechanism of STA on T cells was correlated with the gene change related to multi-signal transduction pathways. Furthermore, we also provided lines of evidence that STA, distinct from glucocorticoids, did not activate the glucocorticoid receptor. These findings would be beneficial for further understanding the therapeutic effects of S. mucronata in the treatment of autoimmune diseases. It also suggested the potential of the natural steroid STA as the effective candidate compounds for use in the treatment of inflammatory and autoimmune diseases.     

Histamine inhibition of the in vitro induction of cytotoxic T-cell responses

Addition of 10-3 to 10-6 M histamine (H)3 to mixed leukocyte cultures (MLCs) inhibited primary in vitro induction of cytotoxic T lymphocytes (CTLs) specific for either allogeneic or trinitrophenol-modified syngeneic target cells. The use of specific H agonists implicated H2 but not H1 receptor triggering in the mediation of these effects. Unlike in vivo-induced allogenic CTLs, the addition of H to assay culture failed to influence the effector function of in vitro-induced CTLs of either specificity. Kinetic studies showed that this difference might be due to loss of functional H receptors after the initiation of the in vitro MCL, and demonstrated that H interferes with an early event in the generation of CTLs. These data indicate that H receptors are not merely markers for CTL precursors, but that they are functional receptors, and suggest that H may play an important role in regulating both the generation and effector function of CTLs in vivo.     

Enhancement of murine thymocyte cytotoxic T cell responses by thymosin

Incubation of murine thymocytes with thymosin Fraction 5 (F5) results in a twofold enhancement of the cytotoxic T lymphocyte response (CTL). The assay exhibits requirements for optimal concentrations of thymosin (100 micrograms/ml) and optimal responder/stimulator ratios. Enhancement of CTL activity can be demonstrated in several responder/stimulator strain combinations. The data indicate that thymosin F5 acts via the responder thymocyte population rather than the stimulator cells, since comparable effects were obtained using nude spleen stimulator cells devoid of mature T cells. This system provides a useful bioassay for identifying the component peptides of thymosin F5 which promote thymocyte differentiation and/or maturation and for elucidating the mechanisms of action of the biologically active thymosin peptides.     

3'-Azido-3'-deoxythymidine inhibition of human lymphocyte cytolytic function in vitro

Despite administration of 3'-azido-3'-deoxythymidine (AZT, Zidovudine) to seriously immunocompromised patients, little has been reported regarding effects of AZT on specific immune functions. This study analyzed the in vitro effect of AZT on normal human lymphocyte cytolytic activity. AZT at concentrations up to 100 microM had no effect when added directly to cytotoxicity assays with lymphocyte effector cells and natural killer (NK)-sensitive or NK-resistant target cells. In contrast, addition of AZT to lymphocytes cultured for 4-10 days with interleukin-2 (IL-2) prior to cytotoxicity assays produced a concentration- and time-dependent inhibition; this effect was not mimicked by acyclovir or ganciclovir. Lymphocyte cell numbers and viability were not reduced in parallel to inhibition of cytolytic activity by AZT. Furthermore, AZT inhibition of IL-2-dependent cytolytic activity was not correlated with alterations in lymphocyte cell surface phenotypes by flow cytometry, and lymphocyte culture supernatant levels of interferon-gamma were not reduced by AZT. These results suggest that AZT may selectively inhibit human lymphocyte functions and thus may have implications for long-term therapeutic administration of AZT in chronic immunodeficiency states.     

Effects of heptaminol AMP amidate on suppressor and helper function of murine T cells

Heptaminol AMP amidate (HAA), a newly developed nucleotide derivative, was found to restore the immunosuppression in mice due to the induction of suppressor T (Ts) cells by concanavalin A (Con A) (50 micrograms/body). HAA also inhibited Con A-mediated in vitro induction of Ts cells. On the contrary, the administration of HAA in mice primed with keyhole lympet hemocyanin (KLH) (30 micrograms/body) caused an enhanced induction of antigen specific helper T (Th) cells. Effects of HAA on Ts and Th cells were found to be dependent on their level of induction. The administration of HAA also increased the spleen cell number and augmented the plaque forming cell response to some extent in cyclophosphamide treated mice. The present results suggested that HAA-mediated immunopotentiation was possible by a combined suppressive effect on Ts cells and enhancing effect on Th cells.     

In vitro and in vivo effects of ketamine on generation and function of dendritic cells

The question about how intravenous anesthetic reagents affect the development and function of dendritic cell subsets still has no comprehensive answers. Bone marrow cells differentiated with FMS-like tyrosine kinase 3 ligand in vitro represented the steady-state dendritic cell subsets. The effects of ketamine on the generation and function of dendritic cell subsets were investigated. We found that dendritic cell subsets responded to the anesthetic reagent ketamine in several aspects: 1) The in vitro and in vivo development of plasmacytoid dendritic cells were inhibited by ketamine at high concentrations; 2) The endocytosis of dendritic cells were not influenced by ketamine at concentrations from 50 - 200 μM; 3) The maturation markers of conventional dendritic cells were not changed by ketamine upon LPS or CpG stimulation, although the cytokines mRNA profiles were affected; 4) The allogenic-stimulatory activity of dendritic cells was suppressed by ketamine. In conclusion, ketamine hampered plasmacytoid dendritic cell subset development both in vivo and in vitro. The dendritic cells maturation and downstream responses towards different toll-like receptor stimuli were differently regulated by ketamine treatment.     

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.     

The relationship between cytotoxic drug exposure and tumour cell kill, in vitro and in vivo.

Doxorubicin has been shown to be more effective against MGH-U1 bladder carcinoma cells grown in monolayer than spheroid. In vitro clonogenic cell survival curves have been replotted against the area under the concentration-time curve (AUC) for drug exposure and fitted to a Hill plot to derive the parameters E max (maximum possible cell kill) and C50 (drug exposure resulting in half the maximum cell kill). The plasma AUC following intraperitoneal administration of doxorubicin to nude mice was measured using a sensitive and specific HPLC assay and combined with the in vitro cell survival parameters to predict the clonogenic cell survival in MGH-U1 xenografts. The Hill parameters from the spheroid model are better predictors of xenograft clonogenic cell survival than the monolayer parameters. It is possible to predict clonogenic cell survival in solid tumours on the basis of the pharmacokinetics of cytotoxic drug exposure, using a mathematical model based on clonogenic cell kill in vitro.     

负载冻融抗原的DC诱导特异性CTL杀伤胃癌细胞的实验研究

目的 研究负载肿瘤抗原的树突状细胞(DC)活化的特异性细胞毒性T淋巴细胞(CTLs)对胃癌细胞的体外杀伤作用.方法 冻融法获取胃癌细胞抗原,联合应用粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)诱导培养外周血BC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs,MTT法检测对胃癌细胞的体外杀伤作用.ELISA法检测γ干扰素(IFNγ)分泌情况.结果 负载胃癌抗原的DC激活的CTLs表现出对胃癌细胞的特异性杀伤作用,产生高水平的IFNY(P<0.01);而对B16黑色素瘤细胞没有杀伤作用不产生高水平的IFNγ.未负载胃癌抗原DC刺激的CTLs对胃癌细胞无杀伤作用.结论 应用细胞因子从人外周血中诱导的DC负载胃癌抗原后,激活的CTLs在体外对胃癌细胞能产生高效而特异性的杀伤作用.     

In vivo and in vitro modulation of MDR molecules in murine thymocytes

P-glycoprotein (Pgp/ABCB1) and multidrug resistance related protein 1 (MRP1/ABCC1) were first described in multidrug resistant tumor cells. It is presently known that both proteins are also expressed in a variety of normal cells, including lymphocytes. ABCB1 activity has already been detected in subpopulations of murine thymocytes, but there was little information on the expression or activity of ABCC1 in these cells. The present work studied in mice the expression of both proteins by RT-PCR and immunofluorescence. It was possible to identify the presence of ABCB1 and to detect the expression of ABCC1 in these cells. The functional activities of these proteins were also studied in vivo and in vitro measuring the extrusion of fluorescent dyes in association with MDR modulators. Cyclosporine A, verapamil and trifluoperazine inhibited the activity of thymic ABCB1. Indomethacin, probenecid and MK571 were effective in inhibiting ABCC1 activity by thymic cells. ABCB1 was only active in a small percentage of thymocytes being present in the immature double negative (not CD4 nor CD8) subpopulation and the mature single positive (CD4 or CD8) subpopulations. The functional activity of ABCC1, on the other hand, was more homogeneously distributed being found in all thymocyte subpopulations. Possible physiological roles for these transporters on thymocytes are discussed.     

Bioactivation of clozapine by murine cardiac tissue in vivo and in vitro

Clozapine, an atypical neuroleptic, undergoes bioactivation to a chemically reactive nitrenium ion. This has been implicated in the pathogenesis of clozapine-induced agranulocytosis. Clozapine also causes myocarditis and cardiomyopathy, the mechanisms of which are unknown. To investigate this, we have evaluated whether clozapine undergoes bioactivation by murine cardiac tissue, in comparison to hepatic tissue. Mice were administered clozapine (5 and 50 mg/kg i.p.), and the extent of covalent binding was assessed by Western blotting. There was an increase in irreversible binding of clozapine to several proteins, ranging in mass from 30 to 250 kDa in both hepatic and cardiac tissue. Bioactivation by hepatic and cardiac microsomes was assessed by LC/MS using glutathione to trap the intermediate. Metabolism of radiolabeled clozapine to a glutathionyl conjugate by liver and cardiac microsomes was 30.5 +/- 3.3 and 3.6 +/- 0.3% of the initial incubation concentration, respectively. Ketoconazole (20 muM), a P450 inhibitor, significantly reduced binding in both hepatic and cardiac microsomes to 6.2 +/- 0.2 and 0.5 +/- 0.06%, respectively. These data indicate that clozapine undergoes bioactivation in the heart to a chemically reactive nitrenium metabolite that may be important in the pathogenesis of myocarditis and cardiomyopathy observed in man.