Morphological alterations in the ovaries of Amblyomma cajennense semi-engorged ticks exposed to ethanolic extract of Acmella oleracea.

The present study analyzed the effects of different concentrations of Acmella oleracea crude ethanolic extract (EEAO) on the development of germ cells from semi-engorged Amblyomma cajennense females in order to evaluate the potential of this natural chemical as a strategy to control these important ectoparasites. A hundred semi-engorged females were divided into five groups (duplicates) (10 animals/group): Control 1 (distilled water); Control 2 (solvent ethanol 50% and DMSO 1%); and Treatment I to III (3.1, 6.2, and 12.5 mg/mL of EEAO, respectively). For the exposure of the ticks to the extract was used the Adult Immersion Test. After the exposition, the ovaries were removed and submitted to histological analysis using Harris hematoxylin and aqueous eosin. The histochemical tests were performed using PAS and Bromophenol blue staining techniques, for the detection of total polysaccharides and total protein, respectively. The extract caused significant alterations in the oocytes, including changes in the shape of the cells, disorganization, and cytoplasmic vacuolation, decrease in the number of yolk granules and germ vesicle fragmentation. These alterations were more intense in the oocytes in initial developmental stages (I and II). The results obtained in this study confirm the cytotoxic potential of the ethanolic extract of A. oleracea on the germ cells of A. cajennense females, opening up the possibility to use this extract as an alternative to control these ectoparasites.

A high throughput screen for next-generation leads targeting malaria parasite transmission.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2-1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male-female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.

In vitro anti-malarial interaction and gametocytocidal activity of cryptolepine.

BACKGROUND: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. METHODS: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. RESULTS: Cryptolepis sanguinolenta (IC50 = 49.65 nM) and its major alkaloid, cryptolepine (IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. CONCLUSIONS: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax).

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.

A new molecular approach for cidal vs static antimalarial determination by quantifying mRNA levels.

In order to maximise compliance, the future antimalarial treatment should ideally require just a single-dose administration. This, in turn, demands new fast-acting effective drugs. Currently, methods to measure the in vitro killing rate of antimalarials are based on parasite growth. We have developed and validated a method to determine and classify antimalarial agents based on their cidal or static activity following quantitative Real Time PCR (RT-PCR) analysis. The method described here is a fast, reliable and user-friendly technique with a medium throughput. Metabolic activity of the parasite is followed by measuring mRNA expression levels of several genes during 5 parasite life cycles. mRNA from the parasite culture is then retrotranscribed to cDNA and quantified by RT-PCR. This new method provides a rapid and reproducible way to accurately measure the antimalarial activity of new compounds in vitro against Plasmodium falciparum.

Protective effect of sodium selenite and zinc sulfate on intensive swimming-induced testicular gamatogenic and steroidogenic disorders in mature male rats.

To investigate the ameliorative potential of sodium selenite and zinc sulfate on intensive-swimming-induced testicular disorders, 48 Wistar male rats (age, 4 months; mass, 146.2 +/- 3.6 g) were randomly divided into 4 groups: the unexercised-control group (n = 12); the exercised group (n = 12); the control supplemented group (n = 12); and the exercised supplemented group (n = 12). For 10 weeks, the exercised rats underwent a protocol that consisted of 4 h.d-1 swimming, for 6 d.week-1; the control rats did not exercise. For 10 weeks, both the supplemented groups received an oral daily dose of a combination of sodium selenite and zinc sulfate (6 and 3 mg.kg body mass-1, respectively). After 10 weeks, a significant reduction (p < 0.05) was seen in rats in the exercised group, compared with rats in both control groups, in paired testicular masses; in epididymal sperm count; in testicular Delta5, 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD; in plasma levels of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin; in the numbers of preleptotine spermatocytes, midpachytene spermatocytes, and stage 7 spermatids of the stage VII seminiferous epithelium cycle; and in fertility performance. As well, a significant increase (p < 0.05) was seen in the exercised group, compared with both control groups, in plasma corticosterone levels and in testicular content of malondialdehyde and catalase activity. At the same time, there was a significant reduction (p < 0.05) in the exercised group, compared with both control groups, in plasma concentrations of zinc and selenium; in the testicular content of glutathione (GSH), the glutathione and glutathione disulphide (GSSG) ratio, ascorbic acid, and alpha-tocopherol; and in testicular activities of superoxide dismutase, glutathione-peroxidase, and glutathione-S-transferase in the testes. No significant changes were seen in the number of spermatogonia-A from the stage VII seminiferous epithelium cycle or the testicular content of GSSG among the groups. Sodium selenite and zinc sulfate supplementation significantly protected against exercise-induced testicular gamatogenic and spermatogenic disorders, prevented testicular oxidative stress, and increased antioxidant status. It can be concluded that intensive-swimming-induced oxidative stress causes dysfunctions in the male reproductive system, which can be protected by the coadministration of sodium selenite and zinc sulfate.

An antimalarial neem leaf extract has both schizonticidal and gametocytocidal activities.

A crude acetone/water (50/50) extract of neem leaves (IRAB) was evaluated for activity against the asexual (trophozoites/schizonts) and the sexual (gametocytes) forms of the malarial parasite, Plasmodium falciparum, in vitro. In separate 72 hour cultures of both asexual parasites and mature gametocytes treated with IRAB (0.5 microg/mL), parasite numbers were less than 50% of the numbers in control cultures, which had 8.0% and 8.5% parasitemia, respectively. In cultures containing 2.5 microg/mL, asexual parasites and mature and immature gametocytes were reduced to 0.1%, 0.2%, and 0% parasitemia, respectively. There were no parasites in the cultures containing 5.0 microg/mL. This extract, if found safe, may provide materials for development of new antimalarial drugs that may be useful both in treatment of malaria as well as the control of its transmission through gametocytes.

Fractions of an antimalarial neem-leaf extract have activities superior to chloroquine, and are gametocytocidal.

The antimalarial activities of two fractions (IRDN-A and IRDN-B) of an extract from the leaves of the neem tree (Azadirachta indica) were compared with those of chloroquine, in in-vitro assays against Plasmodium falciparum. The asexual stages of a chloroquine-sensitive clone (ITG2F6) and a chloroquine-resistant isolate (W2) and the gametocytes of the NF 54 (BD-7) isolate of P. falciparum were used as the drug targets. Activity against the asexual stages was generally evaluated as the concentrations inhibiting the parasitaemias recorded in the control cultures, after an incubation of 48-72 h, by 50% (IC50) or 100% (IC100). For the ITG2F6 strain, the IC50 and IC100 (in microg/ml) were, respectively, 10(-5) and 10(-4) for IRDN-A, 10(-3) and 10(-2) for IRDN-B, and 10(-2) and 1.0 for chloroquine. The corresponding values for the W2 strain were 10(-5) and 1.0 for IRDN-A, and 10.0 and >100 for chloroquine (even at 100 microg/ml, chloroquine only inhibited the parasitaemia by 85%). Each of the two neem-leaf fractions lysed 50% and 100% of developing gametocytes, at 10(-3) and 1.0 microg/ml, respectively; and 50% and 100% of mature gametocytes at 10(-3) and 10(2) microg/ml, respectively. If they are found safe and effective in vivo, the neem-leaf fractions may form the basis of new antimalarial drugs that not only cure chloroquine-sensitive and chloroquine-resistant malaria but also markedly reduce transmission.

Octyl methoxycinnamate: two generation reproduction toxicity in Wistar rats by dietary administration.

Wistar rats continuously received octyl methoxycinnamate (OMC) in the diet through two successive generations at nominal doses of 0, 150, 450 or 1000 mg/kg bw/day. OMC had no adverse effects on estrous cycles, mating behavior, conception, parturition, lactation and weaning, sperm and follicle parameters, macropathology and histopathology of the sexual organs. 1000 mg/kg bw/day reduced parental food consumption and body weight (-14% to -16% in males, -4% to -5% females), increased liver weight, produced hepatic cytoplasmic eosinophilia and erosion/ulceration of glandular stomach mucosa. and led to a slightly decreased implantation rate in the top dose F0 and F1 dams. The high dose F1 and F2 pups had reduced lactation weight gain and organ weights and delayed sexual maturation landmarks. There was no evidence of a selective influence of the test compound on pups' sexual landmarks. The NOAEL (no observed adverse effect level) is 450 mg/kg bw/day for fertility and reproductive performance, for systemic parental and developmental toxicity.

Parasitaemia and gametocytaemia after treatment with chloroquine, pyrimethamine/sulfadoxine, and pyrimethamine/sulfadoxine combined with artesunate in young Gambians with uncomplicated malaria.

As part of a study to assess the infectivity of gametocytes after treatment with four antimalarial regimens, the efficacy of each treatment was also determined. From September to December 1998, 598 children with uncomplicated malaria were treated; 135 received chloroquine (CQ) alone, 276 received pyrimethamine/sulfadoxine (Fansidar, PSD) alone, 113 received PSD with a single dose of artesunate (PSD + 1ART) and 74 received PSD combined with three doses of artesunate (PSD + 3ART). On day 28 19/63 (30.2%; 95% C.I. 19.2% to 43.1%) of children treated with CQ alone, 5/134 (3.7%; 95% C.I. 1.2% to 8.5%) treated with PSD alone, 1/71 (1.4%, 95% C.I. 0.0% to 7.9%) treated with PSD + 1ART and 0/45 (0.0%; 95% C.I. 0.0% to 7.9%) treated with PSD + 3ART were parasitaemic. The proportion of children with gametocytes on day 7 after treatment with CQ alone was 16/89 (18.0%; 95% C.I. 10.6% to 27.6%), 98/174 (56.3%; 95% C.I. 48.6% to 63.8%) after treatment with PSD alone, 8/70 (11.4%; 95% C.I. 5.1% to 21.3%) after treatment with PSD + 1ART and 4/46 (8.7%; 95% C.I., 2.4% to 20.8%) after treatment with PSD + 3ART. CQ thus has a lower efficacy than PSD or either of the PSD and artesunate combinations. Use of PSD alone as an alternative first line treatment results in a very high post-treatment gametocyte prevalence that is likely to enhance transmission. There would be greater and more sustainable benefits from using PSD and artesunate combinations.

Effects of artemisinin derivatives on malaria transmissibility.

BACKGROUND: On the western border of Thailand the efficacy of mefloquine in the treatment of falciparum malaria has declined while gametocyte carriage rates have increased, which suggests increased transmissibility of these resistant infections. We compared the following antimalarial drugs in relation to subsequent Plasmodium falciparum gametocyte carriage: mefloquine, halofantrine, quinine, and the artemisinin derivatives. METHODS: Between 1990 and 1995 we assessed gametocytaemia in a series of prospective studies of antimalarial drug treatment in 5193 adults and children with acute uncomplicated falciparum malaria in an area of malarious hill forest on the western border of Thailand. Weekly parasite counts from thick and thin blood films were done during the 4-week (1990-93) or 9-week (1993-95) follow-up period. Gametocyte positivity rates and person gametocyte week (PGW) rates were calculated to measure gametocyte carriage and transmission potential. FINDINGS: In primary P falciparum infections the gametocyte carriage rate was significantly higher after treatment with mefloquine than after treatment with the artemisinin derivatives (PGW 34.1 [95% CI 25.2-42.9] vs 3.9 [1.9-5.9] per 1000 person weeks; relative risk 8.0 [4.1-15.6]; p<0.0001). Recrudescent infections were associated with increased gametocyte carrier rates (relative risk 2.2 [1.6-3.0]; p<0.0001), but retreatment with artemisinin derivatives reduced subsequent gametocyte carriage 18.5 fold [3.5-98] compared with mefloquine retreatment and 6.8 fold (3.1-15.1) compared with quinine retreatment (p<0.001). The introduction of the artemisinin derivatives in routine treatment at this study site in mid 1994 was associated with a reduction in the subsequent incidence of falciparum malaria of 47 (25-69)% INTERPRETATION: Although environmental changes affect vector numbers, and hence malaria incidence, artemisinin derivatives were found to reduce the transmission potential of falciparum malaria. Widespread introduction of artemisinin derivatives in the treatment of falciparum malaria may prevent the spread of multidrug resistance.

Selenium accumulation, reproductive status, and histopathological changes in environmentally exposed redear sunfish.

Comparisons were made of the accumulation of selenium, histopathological damage, and reproductive status of redear sunfish (Lepomis microlophus) collected in July 1986 from Martin Lake (a contaminated site) and Lake Tyler (a reference site). Hepatic concentrations of selenium were four times higher in Martin Lake sunfish (7.6 +/- 0.5 ppm) than in fish from the reference lake (2.1 +/- 0.2 ppm). Redears collected from the contaminated lake had lower condition factors than individuals collected from the reference site. Sunfish with elevated levels of hepatic selenium had substantial alterations in the liver including necrosis, cytoplasmic vacuolation, and Kupffer cell proliferation. The ovaries of mature fish collected from Martin Lake frequently had atretic follicles, abnormally shaped follicles, connective tissue hypertrophy, asynchronous oocyte development, and an overall reduction in the number of developing oocytes. These histopathological changes in the ovaries of Martin Lake sunfish were not accompanied by alterations in gonadal steroid titers in the blood. No histopathological lesions could be detected in the testes of Martin Lake fish. Most of the males collected from the contaminated site were immature and had lower circulating levels of sex steroid hormones than reference males. The results show that tissue burdens of selenium have declined by 25% since this sunfish population was sampled last in 1981. Further, the results of this study indicate that the overall health and reproductive status of selenium-contaminated fish collected from Martin Lake is still seriously impaired.