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.

Chemical and biological considerations in the treatment of metal intoxications by chelating agents.

Effective chelation treatment of metal intoxications requires that the pharmacokinetics of the administered chelator in fact leads to chelation of the toxic metal, preferably forming a less toxic species which is effectively excreted. This depends on physical and chemical characteristics of metals and chelators as e.g. ionic diameter, ring size and deformability, hardness/softness of electron donors and acceptors, administration route, bioavailability, metabolism, organ and intra/extra cellular compartmentalization, and excretion. In vivo chelation is unlikely to reach equilibrium determined by the standard stability constant, as rate effects and ligand exchange reactions as well as the pharmacokinetics of the chelator considerably influence complex formation. Hydrophilic chelators enhance renal metal excretion, but mainly their extracellular distribution limit their effect to mainly extracellular metal pools. Lipophilic chelators can decrease intracellular stores, but may redistribute toxic metals to e.g. the brain. In chronic metal induced disease, necessitating life-long chelation, toxicity and side effects of the chelator may limit the treatment. The metal selectivity of chelators is important, due to the risk of essential metals depletion. Dimercaptosuccinic acid and dimercaptopropionic sulfonate are presently gaining increased acceptance among clinicians, undoubtedly improving the management of human metal intoxications including lead, arsenic and mercury compounds. Still, development of new safer chelators suited for long-term oral administration for chelation of metal deposits, mainly iron, is an important challenge to the future research.

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.

Localized chrysiasis induced by laser therapy.

BACKGROUND: Chrysiasis is a rare blue-gray skin discoloration that occurs in sun-exposed sites of some patients who receive gold salts. A unique case of localized chrysiasis developed immediately after Q-switched ruby laser (694 nm) irradiation for postinflammatory hyperpigmentation secondary to granuloma faciale in a patient who was receiving long-term gold sodium thiomalate therapy for psoriatic arthritis. Skin biopsy specimens showed striking changes in the ultrastructural characteristics of cutaneous gold deposits following laser treatment. OBSERVATIONS: A blue-gray skin discoloration developed immediately after laser exposure and persisted unchanged after 1 year. Transmission electron microscopy of skin biopsy specimens showed electron-dense gold deposits. Before laser irradiation, these deposits were 106 +/- 35 (mean +/- SD) nm in diameter and faceted, consistent with a crystalline structure. Posttreatment deposits were round, smaller, measured 16 +/- 4 nm, and resembled colloidal gold. Identical findings were observed in an area of sun-protected skin treated with the Q-switched ruby laser; irradiation with a pulsed dye laser at 585 nm had no effect. CONCLUSIONS: Localized chrysiasis was induced in a patient receiving parenteral gold therapy who underwent treatment with a Q-switched ruby laser. This form of chrysiasis resulted from a structural alteration in dermal gold deposits. A similar physiochemical modification in gold deposits induced by UV light may explain the localization of chrysiasis to sun-exposed skin in affected patients.

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.

The utility of chelating agents as antidotes for nephrotoxicity of gold sodium thiomalate in adjuvant-arthritic rats.

The effects of 2,3-dimercaptopropane sulphonate (DMPS) and N-(2-mercapto-2-methylpropanoyl)-L-cysteine (bucillamine) against the renal damage induced by gold sodium thiomalate (AuTM) in adjuvant-arthritic rats were studied. Arthritic rats induced by adjuvant using Mycobacterium butyricum were injected intraperitoneally with a chelating agent (0.6 mmol/kg) immediately after intramuscular injection of AuTM (0.066 mmol/kg) every other day for 21 days. Treatment with DMPS and bucillamine prevented increases in the urinary excretion of protein, aspartate aminotransferase, and glucose and blood urea nitrogen level after AuTM injection. AuTM prevented the increase in both adjuvant-injected and uninjected hind-feet volumes. The prevention of these inflamed lesions by AuTM was not affected by DMPS and bucillamine. These chelating agents decreased the gold concentration in the kidney and liver after AuTM administration, but did not affect the hepatic and renal concentrations of copper, zinc, iron, and calcium except the renal copper level after AuTM. These findings suggest that DMPS and bucillamine are very useful antidotes for gold toxicity.

Cancer morbidity in rheumatoid arthritis patients treated with Proresid or parenteral gold.

The cancer risk was studied by comparison of 305 rheumatoid arthritis (RA) patients exposed to Proresid during a mean time of 22 months and 305 RA patients exposed to sodium aurothiomalate during a mean time of 19 months with the regional cancer register. The mean observation time was 6.9 years (2,117 person-years) for the Proresid-treated and 7.5 years (2,293 person-years) for the gold-treated patients. No increased risk of total malignancies was observed for either group. However, looking at separate tumours, an increased risk of lymphoma and leukemia was found although only significant in the gold-treated group. It was not correlated to dosage or duration of either therapy. The increased risk is consistent with earlier reports of an increased risk of hematopoietic malignancies in RA patients. Marginal over and underreporting, particularly of hematopoietic malignancies, were observed, mainly due to clinicians' failure to report and to recall false reports.

A Case of Remission of Systemic Juvenile Rheumatoid Arthritis(Still's Disease) Treated with High-dose Intravenous Gammaglobulin

High dose intravenous gammaglobuline (IVLG) therapy is effective in some of the autoimmune diseases. Although the exact mechanism of action of IVIG is uncertain, the action as a neutralizing antibody against unknown etiologic agents, the action of blocking of Fc receptors of effector cells, or the action as a antiidiotypic antibody are suggested. We report a case of 12 year old girl with systemic juvenile rheumatoid arthritis who was treated with high dose IVIG and got a remission. In August 1990 she was admitted to our hospital. because of intermittent fever, transient rash and multiple arthralgia. Under the diagnosis of systemic juvenile rheumatoid arthritis, aspirin (4.0g/day) had been given with symptom improvement. She was readmitted in October 1990 because of aspirin intoxication and acute fulminant hepatitis. She was discharged after recovery and any medicine was not prescribed. In November 1990 she was admitted because of epigastric pain, vomiting, intermittent fever, multiple arthritis, and mild hepatomegaly. Total parenteral alimentation had been given under the diagnosis of superior mesenteric artery syndrome and gold sodium thiomalate (Myochrysine, 5 and 10 mg, two weekly IM injection) was given in conjunction with prednisolone (30 mg/day) and naproxen (375 mg/day). She was admitted again in February 1991 due to the fever, coughing, rash, and hepatosplenomegaly. Pneumonia and leukopenia (2100/mm(3)) were found and gold sodium thiomalate injection was discontinued. Gammaglobulin 1 g/kg/day was given intravenously for 2 consecutive days with dramatic symptom improvement. Five more monthly IV gammaglobulin had been given and the side reaction of injection were nausia, fever, and headache which were controlled by the decrease of infusion rate. Four months after the last IVIG injection she had no symtom of arthritis and the hepatosplenomegaly was decreased. Hemoglobin level was increase to 12.2 mg/dL form 6.2mg/dL and ESR was decrease to 15mm/h. The oral prednisolne and ibuprofen were stopped one year after th last IVIG injection. All the laboratory parameters of arthritis and physical examinations had been normal for more than two year after the stop of all the medications until March of 1994. We suggest that high dose intravenous gammaglobulin can be one of treatments for severe systemic juvenile rheumatoid arthritis.

The use of the disease activity score in the analysis of clinical trials in rheumatoid arthritis.

OBJECTIVE: To ascertain how well the disease activity score discriminates drug from placebo treated patients. METHODS: Three placebo controlled trials in rheumatoid arthritis (RA) were reanalyzed using the disease activity score: DAS = 0.53938 x SQRT (Ritchie index) + 0.06465 x (# swollen joints) + 0.330 x 1n (erythrocyte sedimentation rate) + 0.224. RESULTS: Patient groups receiving methotrexate, high dose D-penicillamine and sulfasalazine had the statistically greatest improvement vs placebo treated groups; patient groups receiving gold sodium thiomalate (GSTM) and low dose D-penicillamine also showed statistically significant improvement versus placebo treated groups. Patients receiving sulfasalazine or GSTM were deemed to benefit compared to placebo treated patients in this analysis, unlike the results presented in the initial analyses of this trial. CONCLUSION: The disease activity score is a simple and effective measure of inflammation that can discriminate between active drug and placebo treated patient groups. Use of this composite measure may improve analysis of clinical trials and also be applicable to clinical care.

Which traditional measures should be used in rheumatoid arthritis clinical trials?

It is standard practice to use multiple outcome measures in rheumatoid arthritis (RA) clinical trials. Because of this, multiple testing is usually done, and there is confusion in the interpretation of the results. It is not clear which measures are the most sensitive to detecting improvement and which provide independent information. To address these questions, we conducted an analysis of pooled raw data from 3 placebo-controlled RA trials: one of methotrexate, another of oral and injectable gold, and the third of low-dose and high-dose D-penicillamine. The results show that the joint tenderness count, erythrocyte sedimentation rate, grip strength, and physician assessment of disease activity perform best in RA clinical trials and cover the domain of change measured by traditional outcome measures. Other clinical measures may provide little additional useful information for a standard therapeutic trial of up to 6 months duration, and some measures, such as the proximal interphalangeal joint circumference, hemoglobin level, and 50-foot walking time, can be eliminated from such trials.

[An analysis of the morbid condition in adjuvant arthritic rats and a new method for evaluating anti-rheumatic drugs].

The progress of various symptoms in adjuvant arthritic rats (AA-rats) and the effects of anti-rheumatic drugs were continuously observed on the basis of the Weibull distribution function, and the following results were obtained: 1) The cumulative incidence rates F(t) of various symptoms and abnormalities of measured values gradually increased with the passage of time. The relationship between the F(t) and the days after adjuvant injection indicated a simple Weibull distribution function. On the other hand, the bone damages in the distal limb joints indicated a composite Weibull distribution function with two shape parameters (m1 and m2). It was possible to classify the animals into two groups, fast responders (m1) and slow responders (m2), according to the time of occurrence of bone damages. 2) By using the Weibull probability paper, it was possible to integrate and to evaluate in totality those cases with varying grades of symptoms and cases with different symptoms in the adjuvant-induced syndrome in rats. 3) Azathioprine (AZP) showed an inhibitory effect on the advents of pain and swelling and functional disorders, while indomethacin (IDM), prednisolone (PSL) and gold sodium thiomalate (GST) delayed the advents of these symptoms. As to bone damages in the distal limb joints, IDM, PSL and AZP showed an inhibitory effect in only the fast responder group, while GST showed both an inhibitory and delaying effect in both the fast responder group and the slow one. The above results suggest that the usage of the Weibull distribution function is useful not only for analysis of the morbid condition of AA-rats but also for the evaluation of anti-rheumatic drugs in AA-rats.

Antiarthritic synergism of combined oral and parenteral chrysotherapy. I. Studies in adjuvant-induced arthritis in rats.

In comparative clinical studies of auranofin (AF, oral gold) and parenteral gold in the treatment of rheumatoid arthritis, no difference in efficacy was detected. Since the pharmacologic profiles of these compounds are different, we studied their combined effect on adjuvant arthritis (AA). The effect of AF alone and combined with gold sodium thiomalate (GTM) or gold sodium thiosulfate (GTS) on the excretion of urinary hydroxyproline (UHP) and urinary calcium (UCa), and the articular index of arthritic rats was followed during five weeks of treatment. The excretion of UHP and UCa was significantly inhibited (P less than 0.005) in rats treated with AF combined with GTM or GTS as compared with animals treated with the individual gold compounds. However, the articular index only decreased significantly (P less than 0.02) in the group of rats treated with AF + GTS. The present studies open the possibility that combined treatment with oral and injectable gold provide a new approach for chrysotherapy with an increased antiarthritic potency.

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.

Pharmacological studies of antigen-induced arthritis in BALB/c mice. II. The effects of second-line antirheumatic drugs and cytotoxic agents on the histopathological changes.

The effects of treatment with second-line antirheumatic drugs and cytotoxic agents on the severity of experimental monoarticular arthritis in BALB/c mice have been investigated. The arthritis was assessed histologically in terms of synovitis and erosions of cartilage and bone. Azathioprine (20 mg/kg) and sulphasalazine (10-30 mg/kg oral; 30-100 mg/kg i.p.) produced significant suppression of synovitis and erosions when administered daily for 4 weeks commencing 2 weeks after induction of the arthritis. Dapsone (1-10 mg/kg) and to a lesser extent methotrexate (2 mg/kg) produced some suppression of erosive disease when administered daily for 4 weeks commencing 2 weeks after induction of the arthritis but this failed to reach statistical significance. Chloroquine, D-penicillamine and sodium aurothiomalate all failed to have any effect on the disease with any of the treatment schedules used. Auranofin had no effect on the disease when treatment commenced 2 weeks after intra-articular injection but produced variable suppression at high doses when administered from the day of intra-articular injection.

Epidermolysis bullosa acquisita: clinical response to plasma exchange therapy and circulating anti-basement membrane zone antibody titer.

Epidermolysis bullosa acquisita has been recognized as a rare autoimmune mechanobullous disorder since the detection of immunoglobulin and complement deposits along the basement membrane zone. A circulating anti-basement membrane zone antibody has also been detected in some cases. We are reporting a case of epidermolysis bullosa acquisita in which clinical symptoms were well correlated with the circulating anti-basement membrane zone antibody titers. Although the patient initially responded very well to corticosteroid therapy, remission could not be maintained without increasing the dosage. Other therapies, including azathioprine, dapsone, vitamin E, and gold sodium thiomalate, produced no beneficial effects. Although a high dose of oral corticosteroid and cyclophosphamide decreased the antibody titer and blister formation, this therapy had to be terminated because of side effects. Plasma exchange therapy in combination with corticosteroid and low-dose cyclophosphamide resulted in a marked decrease of the anti-basement membrane zone antibody titer and clinical improvement. Thus plasma exchange therapy may be a useful adjunct to conventional treatments for patients with epidermolysis bullosa acquisita.

Radiographic temporomandibular joint abnormalities in patients with juvenile chronic arthritis during a controlled study of sodium aurothiomalate and D-penicillamine.

Sixty-two patients with juvenile chronic arthritis (JCA) were randomized to 50-week treatment with either sodium aurothiomalate (G) or D-penicillamine (PEN) and followed with regard to radiographic changes of the temporomandibular joints (TMJ). The radiographic methods comprised panoramic radiography and oblique lateral transcranial radiography, occasionally supplemented with other methods. The radiographs were read by a single assessor who did not know the type of drug treatment. Radiographic progression was seen in one G-treated and six PEN-treated patients with polyarticular JCA, and in one G-treated patient with pauci-articular JCA. A relation between changes in radiographic TMJ findings and disease activity was demonstrated. Radiographic examination of the TMJ seems to be of value in therapeutic trials in patients with JCA.

[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.