Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Gold sodium thiomalate improves membrane potential impaired by high-frequency stimulation.

Effects of gold sodium thiomalate (GSTM) on membrane potential and tetanus tension were examined to elucidate whether the gold compound improves mechanical and electrical muscle dysfunction produced by continuous repeated stimulation of frog skeletal muscles. Continuous stimulation (50 Hz for 2 min, 0.05 ms pulse duration) to the sartorius muscle depolarized the membrane, decreased action potential amplitude, and prolonged action potential duration. GSTM (0.1 mM), unlike thiomalic acid (0.1 mM), markedly decreased impairment of these electrical parameters produced during the stimulation period. In the presence of 500 units/mL of catalase, fatigue stimulation still lengthened by 1.5-fold the half-duration of the action potential after a 5-min rest. The prolongation was, however, smaller than that in controls (no catalase). Application of both catalase and GSTM led to no further changes in action potential compared with the application of catalase alone. GSTM did not affect resting tension of single toe muscle fibers though it suppressed the maximum tension after continuous stimulation. These findings suggest that GSTM can inhibit excitable dysfunction of skeletal muscles subjected to continuous stimulation and that such protective effects of GSTM may be partially mediated by H2O2.

Bacterial lipopolysaccharide increases prostaglandin production by rat astrocytes via inducible cyclo-oxygenase: evidence for the involvement of nuclear factor kappaB.

This study was set to investigate the mechanisms through which bacterial lipopolysaccharide (LPS) stimulates prostaglandin (PG) production in rat astrocytes. Primary cultures of rat hypothalamic astrocytes were established. Cells were treated with LPS alone or LPS plus antagonists of various pathways, and the subsequent changes in cyclo-oxygenase (COX) activity were monitored by measuring a COX end product, PGE2, released into the incubation medium. It was found that (i) LPS produced a concentration-dependent increase in PGE2 release from astrocytes. The potency of LPS was significantly increased by the addition of serum into the incubation medium; (ii) after 24 h of incubation, inducible COX (COX-2) accounts for most of the LPS-stimulated PG production, as the latter was markedly reduced by dexamethasone and the specific COX-2 inhibitor NS 398; and (iii) nuclear factor kappaB appears to play a role in the activation of COX-2 induced by LPS, since certain inhibitors of this transcription factor were able to antagonize, at least in part, the effects of LPS on PGE2 release.

Zinc ions antagonize the inhibitory effect of aurothiomalate on glucocorticoid receptor function at physiological concentrations.

The water-soluble gold preparation aurothiomalate, which contains gold as Au(I), is frequently prescribed for patients with rheumatoid arthritis as a disease-modifying agent. We report that aurothiomalate negatively modulates glucocorticoid hormone action; it represses the ligand- and DNA-binding activities and the transactivation function of the glucocorticoid receptor. We suggested the existence of endogenous titrating activities of Au(I) because otherwise administration of aurothiomalate to a patient with rheumatoid arthritis would be expected to result in peripheral insensitivity to glucocorticoids and worsen the patient's status. Focusing on metal ions that are present in vivo, we found that Zn(II) counteracts the inhibitory effect of Au(I) on glucocorticoid receptor function. This complementary effect of Zn(II) was observed at physiological concentrations. We suggest that Zn(II) preserves glucocorticoid receptor function in target tissues and maintains hormone responsiveness, even with chrysotherapy.

Alteration of interleukin-1 activity and the acute phase response in adjuvant arthritic rats treated with disease modifying antirheumatic drugs.

Interleukin-1 (IL-1) activity and the acute phase response, as measured by plasma CRP and iron, were used to determine if the standard disease modifying antirheumatic drugs (DMARDs), gold, chloroquine and D-penicillamine had a common profile of activity in the adjuvant arthritic (AA) rat. All drugs were tested at a dose which significantly reduced noninjected paw swelling in AA rats. Inhibition of paw edema ranged from 37% for D-penicillamine (100 mg/kg) to 69% for auranofin (10 mg/kg). Two week medication of AA rats with gold sodium thiomalate (GST, 10 mg/kg, i.m.) or auranofin (10 mg/kg, p.o.) resulted in a significant decrease in splenic IL-1 activity, as measured in the standard lymphocyte activating factor (LAF) assay. The acute phase response, often associated with elevated IL-1 activity, was also significantly reduced following treatment of AA rats with 10 mg/kg of GST or auranofin (oral gold). Inhibition of the acute phase response by gold was determined by a significant reduction of plasma CRP levels (56-71% reduction) and enhancement of plasma iron levels (27-52% enhancement). In contrast to the effect of GST and auranofin on IL-1, CRP and iron, treatment with chloroquine (20, 30 and 35 mg/kg) and D-penicillamine (55 and 100 mg/kg) failed to reduce the acute phase response (as measured by plasma CRP and iron) or alter LAF activity from AA rat spleen cell supernatants. Based on its ability to reduce LAF activity in spleen cell supernatants and reduce the acute phase response, it is possible that the activity of gold in the AA rat may in part be due to its ability to inhibit IL-1 production in vivo. The inability of chloroquine and D-penicillamine to alter LAF activity and the acute phase response in AA rats does not preclude their possession of an immunoregulatory mechanism of action, but it does indicate that their mechanism of action in the AA rat probably differs from that of GST and auranofin.

Effect of gold salt treatment on the receptor binding activity of monocytes and macrophages isolated from rats with adjuvant arthritis.

We investigated the effect of chrysotherapy on the Fc and complement receptor binding activity of peripheral blood (PB) monocytes and peritoneal (PE) macrophages isolated from normal rats and rats with adjuvant induced arthritis. The adjuvant induced severe disease in Dark Agouti (DA) rats and less marked disease in J. C. Lewis (JC) rats. Gold treatment reduced the disease in DA rats but exacerbated the disease in JC rats. PB monocytes generally exhibited increased receptor activity after adjuvant injection. Gold treatment resulted in a simultaneous reduction of the PB monocyte receptor activity and increased the PE macrophage receptor activity. This was considered to be due to a direct effect of gold, since the Fc receptor activity of PE macrophages increased after in vitro gold treatment.

[The anti-inflammatory effect of auranofin].

The anti-inflammatory effects of auranofin were studied and compared with those of indomethacin, gold sodium thiomalate (GST) and D-penicillamine. Auranofin was active as indomethacin in inhibiting carrageenan induced paw edema in rats, but was less potent than indomethacin in inhibiting UV-induced erythema in guinea pigs. Auranofin inhibited Arthus type paw edema and reverse PCA reaction in rats, on which indomethacin was ineffective. The inhibitory activity of auranofin on adjuvant arthritis was weaker than that of indomethacin. In in vitro experiments, auranofin did not show any suppression of cyclooxygenase activity, but was capable of suppression of lysosomal enzyme release and chemotaxis of neutrophils and macrophages. In addition to these anti-inflammatory activities, auranofin had almost equal anti-analgesic and anti-pyretic activity to that of indomethacin. The above results indicated that the anti-inflammatory profiles of auranofin and indomethacin differ, so we can expect new therapeutic activities of auranofin. GST had similar anti-inflammatory and anti-analgesic profiles to those of auranofin; however, the activities were less potent than auranofin and devoid of anti-pyretic activity. D-penicillamine did not show any anti-inflammatory, anti-analgesic or anti-pyretic activity.

Studies of D-penicillamine on strain variability and lymph node cellularity in adjuvant arthritis.

Experiments were performed in order to ascertain the action of D-penicillamine (PA) on adjuvant arthritis (AA) in rats and to develop a quantitative evaluation method for PA-like drugs, using Lewis, SD, and Wistar rats. Rats were inoculated in the tail with 0.6 mg of Mycobacterium butyricum suspended in 0.1 ml of liquid paraffin. PA apparently induced enhancement of arthritis only in Lewis rats with a good reproducibility. The enhancing effect of PA was seen when it was administered during the period from day -7 to day -1, from day 0 to day 6, or from day 14 to day 20. In control group of Lewis and Wistar rats, adjuvant caused a rapid increase in the cell number of lymph nodes just after the inoculation, and also a marked increase in spleen cells coinciding with the development of arthritis. In PA-treated Lewis rats, the cell numbers of lymph nodes and spleen significantly surpassed those of control rats. However, PA induced no difference from control in Wistar rats, which were not sensitive to PA treatment during the course of arthritis. These results indicate that AA in Lewis rats is a good model for evaluating the activities of PA-like drugs and that PA may affect lymphocytes in lymph nodes and spleen and induce severer arthritis in Lewis rats.

Effects of D-penicillamine, dichlofenac sodium and gold sodium thiomalate upon the selective release of lysosomal enzymes from human polymorphonuclear leucocytes to immune complex.

The selective release of beta-glucuronidase (beta-Gluc) and beta-N-acetylglucosaminidase (beta-Glm) from human polymorphonuclear leucocytes (PMN), initiated with bovine serum albumin/anti-bovine serum albumin (BSA/anti-BSA) immune complex (15 micrograms/ml-1) was significantly reduced by increasing concentrations (10(-7) M, 10(-6) M and 10(-5) M) of D-penicillamine (D-PEN) in a dose-dependent fashion. These effects upon the exocytosis of the lysosomal enzymes studied are in accordance with the results obtained previously in rats with adjuvant arthritis. In contrast, Dichlofenac Sodium (DICHL), which has been found to exert inhibitory activity upon extracellular release of beta-Gluc and beta-Glm in adjuvant arthritic rats in previous studies, had no significant in vitro effect on the exocytosis of these enzymes at the concentrations identical to those of D-PEN. Also, Gold Sodium Thiomalate (GST), in the same concentrations ranging from 10(-7) M-10(-5) M, failed to inhibit selective release of beta-Gluc and beta-Glm in the present investigations. Additionally, BSA/anti-BSA, D-PEN, DICHL and GST did not significantly produce the extracellular release of lactate dehydrogenase (LDH) indicating that under experimental conditions described the cell remained intact. Moreover, neither D-PEN, DICHL, GST or BSA/anti-BSA significantly changed the activities of lysosomal enzyme markers used in these experiments. The possible mechanism(s) of the observed phenomena are discussed.

Effect of antirheumatic agents on the mitogen response of arthritic rat spleen cells.

Splenic lymphoid cells from rats with adjuvant induced polyarthritis show a diminished response to the T-cell mitogens Con A and PHA. This report describes the in vitro effects of various antirheumatic agents on the mitogen induced proliferative response of normal (NSC) and arthritic (ASC) rat spleen cells. Indomethacin enhanced only the arthritic responses. Other PG synthesis inhibitors such as ibuprofen and naproxen enhanced the arthritic as well as the normal spleen cell proliferative responses. Gold sodium thiomalate augmented arthritic but not normal blastogenesis. Penicillamine significantly enhanced only the Con A response of arthritic cells at 10(7)M. Levamisole produced a significant increase in the PHA response of arthritic cells and the Con A NSC response. Chloroquine diphosphate enhanced the Con A response of normal cells at 10(-5)M and 10(-6)M; both chloroquine and tilorone suppressed blastogenesis of arthritic spleen cells at 10(-4)M and 10(-5)M. When classifying antirheumatic agents as stimulator or suppressors of immune function, one must account for their effects on arthritic as well as normal lymphoid cells at the predicted pharmacologic plasma levels.

The influence of diethyl-dithiocarbamate ('Imuthiol') on mononuclear cells in vitro.

Diethyl-dithiocarbamate ('Imuthiol') has been shown to enhance various immune functions in vivo but is toxic in vitro. Macrophages were observed to show evidence of toxicity when exposed to lower concentrations of Imuthiol than inhibited thymidine incorporation by the Raji lymphoid cell line. Inhibition of the mitogenic response of human mononuclear cells to phytohaemaglutinin (PHA) occurred after preincubation of adherent or non-adherent mononuclear cells with Imuthiol. This finding contrasts with the results with gold salts where preincubation of adherent cells inhibits the response to mitogens, while preincubation of non-adherent cells has no effect. The specific toxicity of gold on monocyte/macrophages in vitro is not a feature of Imuthiol.

Biologic actions and pharmacokinetic studies of auranofin.

The preclinical profiles of auranofin (Ridaura), an oral chrysotherapeutic agent, parenteral gold sodium thiomalate, gold thioglucose, and their respective ligands were compared. Auranofin was more effective than gold sodium thiomalate in suppressing inflammation and stimulating cell-mediated immunity. In contrast to gold sodium thiomalate and gold thioglucose, auranofin inhibited cellular release of lysosomal enzymes, antibody-dependent cellular cytotoxicity, production of antibodies in adjuvant arthritic rats, and antibodies involved in cytotoxicity reactions. The respective ligands were without significant biologic activity. In rats, a higher fraction of gold was associated with blood cells after auranofin administration than after gold sodium thiomalate. The absorption, distribution, metabolism, and excretion of auranofin are uniquely different from other gold compounds.

The effect of some antirheumatic drugs in vivo on the response of spleen cells to concanavalin A in rats with chronic inflammation.

During the course of adjuvant arthritis in rats adherent spleen cells inhibited the response of spleen lymphocytes to the T-cell mitogen concanavalin A (Con A). The effects of 14 days treatment with various antirheumatic drugs on spleen cell responsiveness to Con A were investigated. Two nonsteroidal anti-inflammatory drugs, indomethacin (1 mg/kg/day p.o.) and acetylsalicylic acid (200 mg/kg/day p.o.) did not modify the spleen cell response, whereas treatment with chloroquine (50 mg/kg/day p.o.) or levamisole (5 mg/kg/day p.o.) further increased the inhibitory effects of the adherent suppressive spleen cells. On the contrary, treatment with sodium aurothiomalate (10 mg/kg/day i.m.), D-penicillamine (50 mg/kg/day p.o.) or pyritinol (50 mg/kg/day p.o.) significantly enhanced the response of the lymphocytes to Con A. In addition to the effects on spleen cell responsiveness, the ability of the various drug treatments to modify the polyarthritic lesions of the disease was investigated. It is suggested that this model may provide a valuable approach for evaluating the effects of antirheumatic drugs in vivo on immunological responsiveness during chronic inflammatory disease.

Long-term effects of myochrysine on the synovial membrane and aurosomes.

Mysochrysine injected into the rabbit knee joint produced regressive and destructive changes in the synovial membrane, ultimately leading to fibrosis. Aurosomes, containing characteristic electron-dense deposits indicating the presence of gold, formed in the synovial intimal cells and subsynovial macrophages. The number of aurosomes decreased with the passage of time but some were found even 2 yr after the injection of Myochrysine. Electron-probe X-ray analysis showed that the aurosomes contain gold, sulphur and phosphorus. A comparison was made between the atomic ratios of these elements in 3-day and 18-mth-old aurosomes but no significant difference was detected.

Influence of anti-rheumatic drugs on human lymphocytes in vitro.

UNLABELLED: Influence of anti-rheumatic drugs on human lymphocytes, especially T and B cell membranes, was studies with D-penicillamine, aurothiomalate, dexamethasone, cyclophosphamide, mitomycin C and aspirin. METHOD: Peripheral blood obtained from five healthy individuals and lymphocytes were separated by centrifugation with Lymphoprep. The separated lymphocytes were adjusted to 5 X 10(6)/ml in PBS. The suspension of lymphocytes was mixed with equal volume of each concentration of the above drugs. After suspensions, we investigated the percentages of T -and B-cells, compared to control. The results are as follows: 1. Drugs which act only on the T cell membrane: D-penicillamine, aurothiomalate. 2. Drug which acts only on B cell membrane: dexamethasone. 3. Drugs which act on both T- and B-cell membrane: mitomycin C, cyclophosphamide and aspirin.