Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells

The microtubule inhibitor vinblastine causes accumulation of autophagic vacuoles in many cell types. In hepatocytes, many of the accumulated vacuoles are nascent, which has been interpreted to suggest that vinblastine acts by inhibiting the fusion of hydrolase-containing lysosomes with early autophagic vacuoles. However, our previous results suggested that, in Ehrlich ascites cells, vinblastine causes accumulation mainly of older autophagic vacuoles (AVs). This study was undertaken to further characterize the mode of action of vinblastine in these cells. The vinblastine-accumulated AVs were quantified by electron-microscopic morphometry. In addition, the effects of inhibitors of autophagic segregation (leucine, histidine, and 3-methyladenine) on the vinblastine-induced accumulation of autophagic vacuoles were studied. Protein degradation was measured using [14C]valine. Vinblastine caused accumulation of advanced autophagic vacuoles but did not increase the rate of protein degradation. The volume density of early vacuoles remained at the control level. The amino acids retarded but did not prevent the accumulation of autophagic vacuoles, whereas 3-methyladenine almost completely prevented the accumulation. The results suggest that in Ehrlich ascites cells vinblastine acts by inhibiting the maturation of advanced autophagic vacuoles into residual bodies and by stimulating the formation of new autophagic vacuoles. However, 3-methyladenine almost completely prevents the formation of new autophagic vacuoles in the presence of vinblastine. In conclusion, in Ehrlich ascites cells, vinblastine does not prevent the entry of hydrolases into autophagic vacuoles. This calls into question the importance of microtubules in the transport of lysosomal enzymes into autophagic vacuoles.     

Effects of ectopically expressed neuronal Wiskott-Aldrich syndrome protein domains on Rickettsia rickettsii actin-based motility

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling. Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R. rickettsii to assess their effects on rickettsial motility. S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize to R. rickettsii or their actin tails. Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S. flexneri while only moderately inhibiting motility of R. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S. flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R. rickettsii motility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R. rickettsii is independent of N-WASP and the Arp2/3 complex.     

Rhein reduces proteoglycan loss during the autolytic breakdown of cultured cartilage

Rhein (R: 1,8-dihydroxy-3-carboxyanthraquinone) is the active metabolite of the drug diacerhein (DAR), an anthraquinone molecule which has recently been proposed for the long-term treatment of osteoarthrosis. In the present study we have examined the effects of rhein, as compared to indomethacin or hydrocortisone, on an in-vitro model of cartilage degradation, represented by the autolytic breakdown of the articular cartilage excised from rabbit knee and cultured for seven days. During this period there is a spontaneous loss of proteoglycans. At the end of the period we measured the amount of proteoglycans which remained bound to the cartilage. The samples treated with R revealed dose-dependent modifications in the amounts of cartilage-bound proteoglycans, with a 40% increase as compared with non-treated samples at the dose of 7 x 10(-5) M. We conclude that R shows a protective effect on the articular cartilage, and that at least a part of the beneficial effect that DAR has shown in the course of clinical trials in osteoarthrosis may be due to direct effects of its active metabolite (R) on cartilaginous tissue.     

Depression of Human Neutrophil Motility by Influenza Virus In Vitro

After in vitro treatment of human peripheral neutrophils for 60 min at 37°C with purified influenza A virus, their random locomotion (no chemoattractant), chemokinesis (migration in the presence of a chemoattractant without a gradient), and chemotaxis were depressed, as determined by a modification of the Boyden method. The inhibition was accentuated by the presence of human serum devoid of antibodies to the virus. A strain of influenza B virus did not depress neutrophil motility at the same concentration and agglutinated the neutrophils at higher concentrations. Antiserum treatment partly abolished the inhibitory activity of the influenza virus. Inactivation of the virus infectivity with Tween 80 and ether did not change the ability of the virus to inhibit neutrophil motility. Bacterial neuraminidase did not inhibit neutrophil motility. Pretreatment of neutrophils with amantadine hydrochloride, which has been reported to block viral replication in its early stages, did not modify the depression of neutrophil motility by influenza A virus. The results give additional support to the suggested mechanism of depressed neutrophil function as a cause of bacterial superinfection after influenza.     

Effect of amoxycillin and doxycycline on function of human granulocytes tested in vitro and on chemotaxis of granulocytes from rabbits given the two antibiotics

The effect of amoxycillin and doxycyciine on human granulocyte function was studied in vitro. The antimicrobial agents were added to tests of chemotaxis, random motility, yeast phagocytosis, and killing, at progressing concentrations. The effect was also evaluated in vivo by measuring chemotaxis and random motiiity (agarose technique) of granulocytes from rabbits injected with these antibiotics. Amoxycillin was shown to have a slightly stimulating effect on chemotaxis, demonstrable only at the highest concentration tested (100 g/ml), and no effect on the other variables, Doxycyciine had a dose-related inhibitory effect on chemotaxis, random motiiity, and phagocytosis, and no effect on killing. Chemotaxis and random motiiity were slightly, but not significantly, stimulated in vivo when rabbits were given amoxycillin. Chemotaxis (but not random motiiity) was significantly impaired when the rabbits were given doxycyciine.     

Effects of hyaluronan on the invasive properties of human breast cancer cells in vitro

Hyaluronan (HA) is a high molecular weight glycosaminoglycan present mostly in the extracellular matrix (ECM). HA binds to specific receptors such as CD44. Its production is increased at the tumour-stroma interface, including those in breast cancer tumours. It has been suggested that it facilitates invasion of tumour cells into the ECM by a hydrodynamic effect, or by altering tumour cell behaviour. Using in vitro tests we studied the effect of immobilized (iHA) and soluble (sHA) HA on the invasive properties of four human breast cancer cell lines with different levels of CD44 expression. Our results show that iHA acts as an adhesive, haptotactic, and motility stimulating factor for the CD44 positive Hs578T cells and induces the expression of membrane CD44. sHA also changes the motility properties of the Hs578T and MDA-231 cells and increases their CD44 expression. sHA or iHA have no measurable effect on the adhesion, motility or CD44 expression of the ZR-75-1 and MCF-7 breast cancer cells. Our results establish that in high CD44 expressing breast cancer cells HA modulates tumour cell adhesion and motility and also increases the expression of its own receptor, CD44.     

Inhibition of Certain Deoxyribonucleic Acid Viruses by Liver Extracts

A proteinaceous substance was detected in the liver of several animal species that inhibited the multiplication of certain deoxyribonucleic acid, but not ribonucleic acid, viruses. Preliminary experiments suggested that its site of action was not extracellular. It did not inhibit virus by interfering with adsorption to cells. It was suggested that this inhibiting substance acts directly upon cells by interfering with viral synthesis.     

Modulation of non-specific bronchial reactivity

There are several drugs that can modify non-specific reactivity induced by various stimuli. Disodium cromoglycate (DSCG) is able to reduce bronchial hyperreactivity status, but its mechanism has not yet been demonstrated. It would be expected to exert its activity either by mastocyte stabilization or by other actions that are not related to mastocyte mediator release. It has been suggested that DSCG has a calcium channel blocking activity. Its possible role as a calcium antagonist suggest a general modifying effect of calcium antagonists against hyperreactivity, although their use in therapy cannot be foreseen. In exercise-induced bronchospasm, numerous beta 2-adrenergic drugs have proved more effective than other drugs studied in several investigations. It is unknown whether their effects are to be attributed to a bronchodilator activity or to another mechanism of action. Anticholinergic drugs have a lower protective effect against several stimuli. Theophylline has protective action on histamine bronchospasm which is dependent on the blood levels of theophylline attained. Many drugs currently available can modify bronchial hyperreactivity, but only in a transient way. Several anti-asthmatic drugs cause a decrease of the bronchial hyperreactivity, reducing the release of inflammatory mediators and inhibiting reflex bronchoconstriction. However, at present there are no specific drugs which are able to modify bronchial hyperreactivity with a direct action.     

Effect of inhibition of dynein function and microtubule-altering drugs on AAV2 transduction

Over the past decade, adeno-associated (AAV) virus has emerged as an important vector for gene therapy. As a result, understanding its basic biology, including intracellular trafficking, has become increasingly important. Here, we describe the effect of inhibiting dynein function or altering the state of microtubule polymerization on rAAV2 transduction. Overexpression of dynamitin, resulting in a functional inhibition of the minus-end-directed microtubule motor protein dynein, did not inhibit transduction. Equally, treatment of cells with nocodazole, or concentrations of vinblastine that result in the disruption of microtubules, had no significant effect on transduction. In contrast, high concentrations of Taxol and vinblastine, resulting in microtubule stabilization and the formation of tubulin paracrystals respectively, reduced rAAV2 transduction in a vector-dose-dependent manner. These results demonstrate that AAV2 can infect HeLa cells independently of dynein function or an intact microtubule network.     

Modulation of the induction of interferon formation by calcium chloride

The results of the study confirm earlier reports on the stimulating effect of calcium ions on production of interferon induced by poly(I) X poly(C) and on translation of messenger RNA for interferon. Neutralization of the inhibiting activity of interferon production suppressor present in the cytoplasm of normal cells has first been demonstrated which broadens our concepts on the mechanism of action of calcium ions.     

Effect of cyclic adenosine 3'',5''-monophosphate antagonists on endotoxin-induced inhibition of human neutrophil chemotaxis.

We reported previously that Escherichia coli endotoxin inhibited human neutrophil chemotaxis toward C5a. This effect of endotoxin was antagonized by anti-inflammatory steroids. We now report that dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandin E1, isoproterenol, and cholera toxin also antagonize the suppression of chemotaxis by endotoxin. Each compound inhibited the effect of endotoxin in a dose-dependent fashion. To be effective, each compound except cholera toxin had to be present at the time of endotoxin challenge. Furthermore, propranolol blocked the protective effect of isoproterenol against endotoxin but not the protective effect of dibutyrl cyclic adenosine 3',5'-monophosphate or prostaglandin E1. Dibutyryl cyclic guanosine 3',5'-monophosphate, adenosine 5'-monophosphate, phenylephrine, prostaglandin F2 alpha, and carbachol did not modify the suppression of chemotaxis by endotoxin. Anti-inflammatory steroids and dibutyryl cyclic adenosine 3',5'-monophosphate are thought to stabilize phospholipids in certain cell membranes. This phospholipid-stabilizing action may contribute, at least in part, to the protective effect against endotoxin-mediated suppression of neutrophil chemotaxis.     

Defibrotide in vitro inhibits neutrophil activation by a Ca++-involving mechanism

Defibrotide, a polydeoxyribonucleotide provided with a pro-fibrinolytic and prostacyclin-like activity, was studied as an inhibitor of polymorphonuclear leucocyte activation in vitro. It was found capable of dose-dependently (1-8 X 10(-5) M) inhibiting FMLP-induced activation, as shown by a decrease of enzyme release and free-radical formation (superoxide anion generation and chemiluminescence). A similar inhibiting activity was observed on A23187-induced activation. An increase in extracellular Ca++ concentration significantly prevented the effect of defibrotide on ionophore stimulation. When PMA was employed as stimulating agent, the drug did not show any inhibiting effect. Finally the pre-treatment of cells with theophylline markedly reduced the inhibition by defibrotide of FMLP- and A23187-dependent activation. Since the stimulation of neutrophils by FMLP and A23187 depends on the increase of cytoplasmic free-calcium availability or extracellular calcium entrance respectively, whereas PMA activation is completely independent from any Ca++ change, the inhibiting effect of defibrotide could be attributed to a Ca++-involving mechanism.     

Effect of the new theophylline derivative on leukocytes migration in vitro

New theophylline derivative: 7-beta-hydroxy-benzylopiperazinopropyl-theophylline (R6) has been investigated for its influence on spontaneous migration of leukocytes, as well as for their response to calcium ionophore A23187 and phytohemagglutinin (PHA). Leukocytes from the peripheral blood of healthy donors were used and migration developed according to the routine capillary method. It has been found that R6 inhibited migration most effectively if the cells have not been treated with other agents. An addition of R6 to media containing A23187 has slightly modified an original action exerted by ionophore itself, the final effect being dependent on calcium concentration in the medium. When R6 was applied simultaneously with PHA its effect on leukocyte migration was negligible, if however R6 was added to leukocytes previously pretreated with PHA--an ultimate increase in migration inhibition was observed. It is suggested that R6 inhibits both spontaneous locomotion of leukocytes and their response to stimulating agents acting via an increase in intracellular cAMP level and/or by hindering the calcium transport across cell membrane.     

Inhibition of differentiation in a murine F9 embryonal carcinoma cell subline by leukemia inhibitory factor (LIF).

Leukemia inhibitory factor (LIF) is a cytokine previously shown to maintain pluripotent embryonic stem cells in their undifferentiated state. We have examined the effects of LIF in nullipotent embryonal carcinoma cell lines, and have found that LIF blocks differentiation induced by retinoic acid and at low temperature in OTF9 cells. LIF did not block differentiation in a parent F9 cell line. For OTF9 cells, LIF acts early in differentiation, inhibiting the appearance of parietal endoderm-type product cells. However, it acts subsequent to retinoic acid, and at least one early retinoic acid-induced event is unaltered in the presence of LIF. This finding provides both a means of dissecting the cascade of events leading to EC cell differentiation, and a well-characterised target cell type for studying the mechanism of action of LIF.     

Inhibitory effect of cetirizine 2HCl on eosinophil migration in vivo

The effect of a potent antihistamine, cetirizine, was studied on allergic patients and normal subjects by means of an in-vivo 'skin window' technique. All subjects showed significant inhibition of skin-test responses to grass pollen, compound 48/80, histamine and methacholine, after administration of a single dose (10 mg) of cetirizine. Compared to placebo, cetirizine significantly decreased the eosinophils attraction at skin sites challenged with grass pollen and compound 48/80. In allergic patients no change in eosinophil migration pattern was noted with histamine and methacholine skin-tested sites. In normal subjects, compound 48/80 and histamine did not induce eosinophil accumulation and cetirizine did not modify cellular patterns as compared to placebo. These results suggest that cetirizine acts on eosinophil migration by inhibiting the release of mast cell mediators or inhibiting the eosinophilotactic mediators themselves.     

Effects of cilostazol,a selective cyclic AMP phosphodiesterase inhibitor on isolated rabbit spinal arterioles

Cilostazol, a potent inhibitor of guanosine 3':5'-cyclic monophosphate (cGMP)-inhibited adenosine 3':5'-cyclic monophosphate (cAMP) phosphodiesterase (PDE3), has been used clinically for the treatment of chronic peripheral arterial occlusive disease. The beneficial effect of cilostazol is attributed to both anti-platelet aggregating activity and vasodilation. However, the effect of cilostazol on resistance-sized vasculature is not well documented. Furthermore, mechanisms of vasodilation and influence on endothelium function are not fully understood. Thus, we investigated the vasodilator action of cilostazol using isolated, pressurized rabbit spinal arterioles with special reference to the functional endothelium. Cilostazol, acetylcholine (ACh), isocarbacyclin (prostacyclin analogue), and sodium nitroprusside (SNP) all produced concentration-dependent vasodilations of isolated spinal arterioles with endogenous myogenic tone. The order of potency of these agonists was isocarbacyclin>ACh>SNP>cilostazol. Indomethacin (10 micro M, a cyclo-oxygenase inhibitor), N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 30 micro M), or chemical denudation of the endothelial cells did not significantly alter the cilostazol-induced arteriolar dilation. Furthermore, stimulating the release of endothelium-derived relaxing factors by administering ACh (100 nM), or treating with isocarbacyclin (1 nM) or SNP (3 nM) did not significantly modify the cilostazol-induced vasodilation. These results suggest that cilostazol produces the vasodilation of isolated, pressurized rabbit spinal arterioles independent of the functional endothelium. We infer that the vasodilator action of cilostazol in the spinal arterioles may be attributed to a yet unknown mechanism that is independent of the PDE3 inhibition.     

Modulation of human immune response by Echinococcus granulosus antigen B and its possible role in evading host defenses

By directly suppressing the function of certain immune cell subsets and by stimulating other cell populations related to immunopathology, parasite-derived substances play an important role in the chronic establishment of parasitic disease. Our objective was twofold: (i) to investigate further the role of Echinococcus granulosus antigen B (AgB) in the human early inflammatory response by determining its effect on polymorphonuclear cell (PMN) random migration, chemotaxis, and oxidative metabolism and (ii) to determine its action in acquired immunity by evaluating AgB and sheep hydatid fluid (SHF)-driven Th1 (gamma interferon [IFN-gamma] and interleukin 12 [IL-12]) and Th2 (IL-4 and IL-13) cytokine production by peripheral blood mononuclear cells (PBMC) from 40 patients who had cured or stable or progressive cystic echinococcosis. AgB significantly inhibited PMN recruitment but left their random migration and oxidative metabolism unchanged. Patients' PBMC stimulated with AgB produced IL-4 and IL-13 but did not produce IL-12. They also produced significantly lower IFN-gamma concentrations than did PBMC stimulated with SHF (P = 10(-5)). AgB skewed the Th1/Th2 cytokine ratios towards a preferentially immunopathology-associated Th2 polarization, predominantly in patients with progressive disease. AgB-stimulated patients' PBMC also proliferated less than SHF-stimulated PBMC (P = 9 x 10(-3)). In vitro Th2 cytokine production was reflected in vivo by elevated specific immunoglobulin E (IgE) and IgG4 antibodies binding to AgB. These findings confirm that AgB plays a role in the escape from early immunity by inhibiting PMN chemotaxis. They also add new information on the host-parasite relationship, suggesting that AgB exploits the activation of T helper cells by eliciting a nonprotective Th2 cell response.     

Effects of caerulein on the gastric motility of rats

The effects of caerulein on gastric motility in urethane-anesthetized rats were studied. Caerulein administered into the lateral cerebral ventricle (i.c.v.) and jugular vein (i.v.) caused predominantly an inhibitory effect on gastric motility but sometimes an excitatory or a biphasic effect. The inhibitory response was reduced after vagotomy and/or splanchnicotomy, or after guanethidine. The remaining inhibitory response was abolished by tetrodotoxin, but was resistant to atropine and guanethidine. The excitatory response was abolished by atropine. Discharges of the gastric branch of the vagus nerve were decreased by i.v. injection of caerulein but increased by i.c.v. injection, whereas those of the splanchnic nerve were increased by both i.v. and i.c.v. injection. These results suggest that caerulein causes an inhibition of gastric motility by centrally stimulating vagal non-adrenergic inhibitory nerves and splanchnic adrenergic nerves and inhibiting vagal cholinergic nerves, and by peripherally stimulating non-adrenergic inhibitory neurons of the myenteric plexus. This peptide causes an excitation by stimulating cholinergic neurons of the myenteric plexus.     

Mechanism of immunosuppressive effect of alprazolam: alprazolam suppresses T-cell proliferation by selectively inhibiting the production of IL2 but not acquisition of IL2 receptor.

The purpose of this study was to elucidate the mechanism of action of alprazolam on concanavalin A (Con A)-induced murine T-cell proliferation. Splenic cells of BALB/c mice were first cultured with an optimum dose of Con A in the presence or absence of varying doses of alprazolam to assess effects of alprazolam on T-cell proliferation, interleukin 2 (IL2) production and IL2 receptor (IL2R) expression. Then, Con A-induced T-blast cells from BALB/c mice were cultured with an excess dose of human recombinant IL2 (rIL2) or crude rat IL2 supernate in the presence or absence of alprazolam to assess the effects of alprazolam on the interaction of IL2 and IL2R. The results of these studies clearly demonstrated that alprazolam can inhibit the T-cell proliferation in response to Con A but not to IL2. Alprazolam also reduced the production of IL2 by splenic T-cells, but did not alter the expression of IL2R on Con A-induced T-blast cells. Furthermore, the results also showed that (a) alprazolam did not inhibit the proliferative response of splenic T-cells to a combination of phorbol 12-myristate-13-acetate (PMA) and ionomycin, and (b) the addition of exogenous IL2 reversed the inhibitory effect of alprazolam on T-cell proliferation. Finally, the addition of alprazolam produced a time-dependent inhibiting effect on T-cell proliferation. However, this inhibitory effect of alprazolam was abolished when the drug was added to the cultures of competent cells that fully expressed IL2R. Taken together, these results suggest that alprazolam inhibits murine T-cell proliferation by affecting the mitogenic receptor-mediated events (initiation) rather than the IL2R-mediated events (progression) of ligand-activated T-cells through the cell cycle.