Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.     

Trypanosoma cruzi: antiprotozoal activity of parthenolide obtained from Tanacetum parthenium (L.) Schultz Bip. (Asteraceae, Compositae) against epimastigote and amastigote forms

This study reports the activity of crude extracts, fractions and parthenolide (pure compound) obtained from Tanacetum parthenium against two forms of the parasite Trypanosoma cruzi. Feverfew is a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis. Activity against epimastigote forms was observed for crude extracts, fractions and parthenolide, and a progressive increase in the antitrypanosomal effect was observed in the course of the purification process. The pure compound showed IC50/96h and IC90/96h of 0.5 microg/ml and 1.25 microg/ml, respectively. The cytotoxic effect of parthenolide in LLMCK2 cells was 3.2 microg/ml (CC50/96h) and the selectivity index was 6.4. No hemolysis was detected for the pure compound. The internalization index of T. cruzi in LLMCK2 cells was reduced almost 51% at the concentration of 2 microg/ml of parthenolide, and 96.6% at 4 microg/ml. Scanning and transmission electron microscopy permitted observation of morphological modifications and ultrastructural alterations.     

Dual effect of heparin on cultured adult rat cardiomyocytes

Heparin has been widely reported to inhibit the growth of several cell types including neonatal rat cardiac myocyte (NRCM) but its effect on adult rat ventricular myocyte (ARVM) is unknown. To determine whether heparin is able to modulate ARVM protein synthesis capacity and if so which pathway is involved in this response, ARVM were cultured in presence or absence of 5% human serum and exposed to heparin (2-2,000 microg/ml) or its analogue xylan (0.5 and 50 microg/ml), and either the Ca(2+) chelator BAPTA/AM (10 microg/ml), or the calcineurin inhibitor FK506 (10 microg/ml), and heparinase I (0.1-10 U/ml) for 2 days. The protein synthesis (PS) was measured after 24 h incorporation of [14C]-Phenylalanine in ARVM. Independently of the serum presence, heparin and xylan altered PS in a bimodal dose-dependent manner. At high doses, heparin and xylan (2,000 and 50 microg/ml, respectively) either had no effect (without serum) or inhibited PS (with serum). In absence of serum, low doses of heparin or xylan (20 and 0.5 microg/ml, respectively) amplified the PS process in ARVM (2-fold, P < 0.05). FK506 inhibited the trophic response to 20 microg/ml heparin alone (-39%, P < 0.05). In presence of serum, the heparin induced-trophic effect, that was not significantly altered by FK506, was inhibited by BAPTA/AM (-32%, P < 0.05). Finally, heparinase I that increased PS in NRCM had no effect on ARVM growth. This study strongly suggests that heparin dose-dependently modulated PS in ARVM, this result being not observed in neonatal cells. Different mechanisms involving intracellular Ca(2+) play a role in the PS response of ARVM to low concentrations of heparin, the intracellular pathways depending on the presence of serum.     

Trypanosoma cruzi epimastigotes: regulation of myo-inositol transport by effectors of protein kinases A and C

Inositol is the precursor for most Trypanosoma cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. Here, we investigated the modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology. Pre-incubation of the cells with either dibutyryl-cyclic AMP (25 microM) or forskolin (30 microM) decreased the myo-inositol uptake by half, this effect being reversed by KT5720 (PKA inhibitor). Conversely, pre-incubation of the cells with PMA (2.8 microg/ml) or serum (5%) had a approximately 50% stimulation in myo-inositol uptake, being this effect reversed by staurosporine (0.5 microM) or sphingosine (10 microM). These results allow us to conclude that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors.     

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.     

Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 10⁴ conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

In vivo effects of Panax ginseng extracts on the cytochrome P450-dependent monooxygenase system in the liver of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed guinea pig

The effects of the subchronic administration of Panax ginseng extracts were examined on the hepatic cytochrome P450-dependent monooxygenase system of guinea pigs pre-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Panax ginseng extracts were intraperitoneally administered to guinea pigs at 100 mg/kg/day for 14 days from 1 week after a single intraperitoneal injection of 1 microg of TCDD/kg of body weight. TCDD treatment increased the total cytochrome P450 content 2.86-fold, and this was remarkably inhibited by the administration of Panax ginseng extracts. Treatment with ginseng extract alone also decreased the contents of cytochrome P450 by 33%, but both TCDD and ginseng extracts had no effect on cytochrome b(5) content. The administration of TCDD resulted in a 1.73-fold increase in microsomal NADPH-cytochrome P450 reductase activity in the guinea pig liver, and this was significantly inhibited by ginseng extracts, but treatment with ginseng extracts alone had no effect on its activity, and no statistical changes in the activity of NADPH-cytochrome b(5) reductase were observed in guinea pig liver due to TCDD and/or ginseng extract administration. Compared to the control, ECOD activity remarkably (1.76-fold) increased after TCDD administration, but this increase was completely inhibited by treatment with ginseng extract. Treatment with ginseng extract alone resulted in a 50% reduction of ECOD activity. TCDD administration remarkably induced benzphetamine demethylation (BPDM) activity, while ginseng extract also slightly increased the enzyme's activity, but the induction attributed to ginseng extracts was not statistically significant. Even though administration of ginseng extracts slightly inhibited TCDD-induced BPDM activity, the inhibition was not statistically significant. These results indicate that ginseng extract exerts different effect on the induction of P450 isozymes. From these results, we suggest that Panax ginseng extracts may act as an inhibitor of CYP1A rather than that of CYP2B.     

Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops

A defined mixture of rho iso-alpha-acids (RIAA), a modified hop extract, was evaluated for anti-inflammatory efficacy and safety. RIAA inhibited LPS-stimulated PGE(2) formation with >200-fold selectivity of COX-2 (IC(50)=1.3 microg/ml) over COX-1 (IC(50)>289 microg/ml). This occurred only when RIAA was added prior to, but not post, LPS stimulation. Consistent with this observation, RIAA produced no physiologically relevant, direct inhibition of COX-1 or COX-2 peroxidase activity. This suggests that RIAA inhibits inducible but not constitutive COX-2. In support, we found RIAA showed minimal PGE(2) inhibition (IC(50)=21mug/ml) relative to celecoxib (IC(50)=0.024 microg/ml), aspirin (IC(50)=0.52 microg/ml) or ibuprofen (IC(50)=0.57 microg/ml) in the AGS gastric mucosal model, where COX-1 and -2 are expressed constitutively. Taken together these results predict RIAA may have lower potential for gastrointestinal and cardiovascular toxicity observed with COX enzyme inhibitors. Following confirmation of bioavailable RIAA administered orally, gastrointestinal safety was assessed using the fecal calprotectin biomarker in a 14-day human clinical study; RIAA (900 mg/day) produced no change compared to naproxen (1000 mg/day), which increased fecal calprotectin 200%. Cardiovascular safety was addressed by PGI-M measurements where RIAA (1000 mg) did not reduce PGI-M or affect the urinary PGI-M/TXB(2) ratio. Drug interaction potential was evaluated against six major CYPs; of relevance, RIAA inhibited CYP2C9. Toxicity was assessed in a 21-day oral, mouse subchronic toxicity study where no dose dependent histopathological effects were noted. Clinically, RIAA (1000 mg/day) produced a 54% reduction in WOMAC Global scores in a 6-week, open-label trial of human subjects exhibiting knee osteoarthritis.     

Effect of Aldicarb,Ethoprop, and Carbofuran on Control of Coffee Root-knot Nematode,Meloidogyne exigua

Egg hatch of Meloidogyne exigua was significantly inhibited in 14 days pretreatment with aldicarb, ethoprop, or carbofnran at concentrations higher than 0.1 μg/ml; these eggs were found to delay hatch in 19 days posttreatment in ethoprop. Aldicarb and carbofuran solutions at concentrations greater than 0.1 μg/ml significantly decreased the motility and the life span of the second-stage juveniles; aldicarb was more toxic than carbofuran to the nematode. In a field test, aldicarb (Temik 10G), ethoprop (Mocap 10G), and carbofuran (Furadan 5G and Furadan Liquid 350F) significantly decreased M. exigua populations.     

Quercus infectoria galls possess antioxidant activity and abrogates oxidative stress-induced functional alterations in murine macrophages

The present study reports the antioxidant activity of ethanolic extract of Quercus infectoria galls. The antioxidant potency of galls was investigated employing several established in vitro model systems. Their protective efficacy on oxidative modulation of murine macrophages was also explored. Gall extract was found to contain a large amount of polyphenols and possess a potent reducing power. HPTLC analysis of the extract suggested it to contain 19.925% tannic acid (TA) and 8.75% gallic acid (GA). The extract potently scavenged free radicals including DPPH (IC(50)~0.5 microg/ml), ABTS (IC(50)~1 microg/ml), hydrogen peroxide (H(2)O(2)) (IC(50)~2.6 microg/ml) and hydroxyl (*OH) radicals (IC(50)~6 microg/ml). Gall extract also chelated metal ions and inhibited Fe(3+) -ascorbate-induced oxidation of protein and peroxidation of lipids. Exposure of rat peritoneal macrophages to tertiary butyl hydroperoxide (tBOOH) induced oxidative stress in them and altered their phagocytic functions. These macrophages showed elevated secretion of lysosomal hydrolases, and attenuated phagocytosis and respiratory burst. Activity of macrophage mannose receptor (MR) also diminished following oxidant exposure. Pretreatment of macrophages with gall extract preserved antioxidant armory near to control values and significantly protected against all the investigated functional mutilations. MTT assay revealed gall extract to enhance percent survival of tBOOH exposed macrophages. These results indicate that Q. infectoria galls possess potent antioxidant activity, when tested both in chemical as well as biological models.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Fasciolagigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether

Objective

To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciolagigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM).

Methods

Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 μg/ml) or both in half concentration of either (5, 10 and 25 μg/ml).

Results

Exposure of Fasciola worms to 25 + 25 μg/ml of combined drug regimens or to 50 μg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin.

Conclusions

Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.     

Role of benzodiazepine-GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice

In the present study, the role of benzodiazepine-GABAA receptor complex in the attenuation of U-50,488H (U50), a selective kappa opioid agonist-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin (GTS) was investigated using the mice tail-flick test. The intraperitoneal (i.p.) treatment of GTS (100 and 200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently attenuated the U50 (40 mg/kg, i.p.)-induced analgesia. GTS (0.001-10 microg/ml) did not alter binding of [3H]naloxone to mice whole brain membrane. The attenuation effect of GTS (100 mg/ kg) and diazepam (0.5 mg/kg) on U50-induced analgesia was blocked by flumazenil (0.1 mg/kg, i.p.), a benzodiazepine receptor antagonist, and picrotoxin (1 mg/kg, i.p.), a GABAA-gated chloride channel blocker. However, bicuculline (1 mg/kg, i.p.), a GABAA receptor antagonist blocked the attenuation effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on U50-induced analgesia. Chronic treatment (day 4-day 6) of GTS (50-200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) on chronic treatment blocked the inhibitory effect of GTS (100 mg/kg) and diazepam (0.5 mg/kg) on tolerance to U50-induced analgesia. On the other hand, chronic treatment of bicuculline (1 mg/kg) blocked the inhibitory effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on tolerance to U50-induced analgesia. In conclusion, the findings suggest that GTS attenuates U50-induced analgesia and inhibits tolerance to its analgesia and this action involves benzodiazepine receptors and GABAA-gated chloride channels.     

Topical Treatment of Dermatophytic Lesion on Mice (<Emphasis Type="Italic">Mus musculus</Emphasis>) Model

Antidermatophytic potential of three weed plants viz. Tridax procumbens L., Capparis decidua (forsk) Edgew and Lantana camara L. were explored and experimentally induced dermatophytic lesion was topically treated in mice. Microbroth dilution method was carried out for determination of MIC and MFC of different extracts of selected plants. In animal studies, mice were experimentally inoculated with Trichophyton mentagrophytes and infected animals were topically treated with 5 mg/g terbinafine and two concentrations, i.e., 5 and 10 mg/g of test extract ointment. Complete recovery from the infection was observed on 12th day of treatment for reference drug terbinafine (5 mg/g) and 10 mg/g concentration of test extract ointment whereas 5 mg/g concentration of test extract ointment showed complete cure on 16th day of treatment. Fungal burden was also calculated by culturing skin scrapings from infected animals of different groups. Test extract ointment successfully treated induced dermatophytosis in mice without any disease recurrence incidences, thereby indicating efficacy of test extract as an excellent topical antifungal agent for the cure of dermatophytosis.     

The mechanisms and costs of physiological and toxicological acclimation to waterborne silver in juvenile rainbow trout (Oncorhynchus mykiss)

Juvenile rainbow trout were exposed to 0, 0.1, 1, 3, and 5 micro g/l silver (Ag, as AgNO3) for 23 days. Specific growth rate, cumulative food consumption, food-conversion efficiency, and critical swimming speed (U(crit)) were significantly reduced during 5 micro g/l Ag exposure, demonstrating a physiological cost of silver acclimation. Only the 5 microg/l Ag treatment had significant cumulative mortality (5.2%). Fish were most susceptible to silver on days 5 and 15. Exposure to 5 microg/l Ag significantly lowered plasma Na+ and Cl- on days 5 and 10, but plasma ion concentration recovered thereafter. Unidirectional Na+ uptake and gill Na/K-ATPase activity were significantly inhibited by 3 and 5 microg/l Ag exposure. Na+ uptake was inhibited by 3 micro g/l Ag at day 5 alone, whereas the effects at the highest Ag exposure persisted until day 15. Gill Na/K-ATPase was inhibited on day 5 in both the 3 and 5 microg/l Ag treatments but increased to approx. 1.5 times of control levels by day 23. Only the 3 and 5 microg/l Ag treatments produced toxicological acclimation (at least twofold elevations in 168-h LC50 values in fish subsampled on days 15 and 23). We conclude that physiological acclimation results from compensatory changes in Na+ transport at the gills, and that these changes may eventually lead to toxicological acclimation.     

Mustard seed meal mixtures: management of Meloidogyne incognita on pepper and potential phytotoxicity

Meals produced when oil is extracted from seeds in the Brassicaceae have been shown to suppress weeds and soilborne pathogens. These seed meals are commonly used individually as soil amendments; the goal of this research was to evaluate seed meal mixes of Brassica juncea (Bj) and Sinapis alba (Sa) against Meloidogyne incognita. Seed meals from Bj 'Pacific Gold' and Sa 'IdaGold' were tested alone and in combinations to determine rates and application times that would suppress M. incognita on pepper (Capsicum annuum) without phytotoxicity. Rates of soil application (% w/w) for the phytotoxicity study were: 0.5 Sa, 0.2 Bj, 0.25 Sa + 0.25 Bj, 0.375 Sa + 0.125 Bj, 0.125 Sa + 0.375 Bj, and 0, applied 0 - 5 weeks before transplant. Overall, 0.2% Bj was the least toxic meal to pepper seedlings. By comparison, 0.5% S. alba seed meal did not reduce lettuce (Lactuca sativa) seed germination at week 0, but all seed meal treatments containing B. juncea prevented or significantly reduced germination at week 0. The seed meals did not affect lettuce seed germination at weeks 1-5, but hypocotyl growth was reduced by all except 0.2% Bj at weeks 1, 4 and 5. Brassica juncea and Sa meals were tested for M. incognita suppression at 0.2, 0.15, 0.1 and 0.05%; mixtures were 0.1% Sa + 0.1% Bj, 0.15% Sa + 0.05% Bj, and 0.05% Sa + 0.15% Bj. All treatments were applied 2 weeks before transplant. The 0.2% Bj and 0.05% Sa + 0.15% Bj treatments overall had the longest shoots and highest fresh weights. Eggs per g root were lowest with 0.1 - 0.2% Bj amendments and the seed meal mixtures. The results indicate that Bj and some Bj + Sa mixtures can be applied close to transplant to suppress M. incognita populations on pepper; consequently, a seed meal mixture could be selected to provide activity against more than one pest or pathogen. For pepper, care should be taken in formulating mixtures so that Sa rates are low compared to Bj.     

A possible involvement of ion transporter in tumor necrosis factor alpha and cycloheximide-induced apoptosis of endothelial cells

We examined the tumor necrosis factor alpha (TNFalpha)-induced apoptosis of vascular endothelial cells from the standpoint of ion channels. Cultured vascular endothelial cells from bovine carotid artery were used. Apoptosis was determined by a propidium iodide assay. Treatment of the endothelial cells with TNFalpha and cycloheximide for 6 h induced nuclear fragmentation in a TNFalpha dose-dependent manner (1-10 ng/ml). Concomitant treatment of endothelial cells with TNFalpha at a dose of 10 ng/ml and cycloheximide at a dose of 10 microg/ml elicited endothelial cell apoptosis as high as 23.4+/-4.1% at 6 h after administration. However, 10 ng/ml TNFalpha alone elicited a little apoptosis at 6 h after its administration (% apoptosis=4.1+/-0.8%). Cycloheximide (10 microg/ml) did not induce apoptosis at all. Concomitant treatment of endothelial cells with 1 mmol/l of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, which is a chloride bicarbonate exchanger blocker, partially inhibited the TNFalpha and cycloheximide-induced endothelial cell apoptosis. On the other hand, endothelial cell apoptosis due to TNFalpha and cycloheximide was completely inhibited by benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (50 micromol/l), an inhibitor of caspase. Moreover, pyrrolidine dithiocarbanate, an inhibitor of nuclear factor kappa B (NF-kappaB), also suppressed endothelial cell apoptosis induced by TNFalpha and cycloheximide completely. These findings suggest that the endothelial cell apoptosis induced by TNFalpha and cycloheximide is closely related to not only chloride ions, but also both NF-kappaB and caspase activation. That is to say, there is a possibility that chloride ions or bicarbonate (pH) may play an important role in signal transduction such as NF-kappaB and caspase activation in the apoptosis induced by TNFalpha and cycloheximide.     

Effects of combined therapy with thalidomide and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 510(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.     

Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (Aminosidine) and gentamicin

Cutaneous leishmaniasis is presently treated with 20 days of parenteral therapy with a frequently toxic drug (antimony). Topical formulations of paromomycin (15%) plus methylbenzethonium chloride (MBCL, 12%) or plus urea (10%) in soft white paraffin have been tested for Old and New World disease in humans. We compared the efficacy of a new topical formulation, WR 279,396 (paromomycin [15%] plus gentamicin [0.5%]) to the clinical formulations in the treatment of cutaneous disease in BALB/c mice. Sixty-day-old lesions were treated twice a day for 10 days, and the response to therapy was determined over a further 70 days. For ulcers due to Leishmania major or to Leishmania mexicana, 100% of lesions in the WR 279,396 group healed by day 20 after therapy and did not relapse by day 70; 83% of lesions healed without relapse in the paromomycin-MBCL group. In the paromomycin-urea group, 100% of L. major lesions healed by day 30 but 30% relapsed. For ulcers due to Leishmania panamensis or Leishmania amazonensis, all lesions treated with WR 279,396 healed and did not relapse; < 50% of lesions treated with paromomycin-MBCL healed by day 30, and all lesions relapsed by day 70. In addition to being active, WR 279,396 was not toxic in this model and appears to have a cosmetic effect (promoting hair growth, healing, and limiting the size of the scar).