Determination of the effective dose rate for monepantel (AAD 1566) against adult gastro-intestinal nematodes in sheep

Monepantel (AAD 1566) is the first compound from the recently discovered amino-acetonitrile derivative (AAD) class of anthelmintics to be developed for use in sheep. Three dose determination studies were conducted in Australia and Europe to identify the therapeutic dose of monepantel, when formulated for the oral treatment of sheep, to control adult gastro-intestinal nematodes. In each study, sheep infected with various nematode species were treated with either 1.25, 2.5 or 5.0 mg monepantel/kg bodyweight. Following euthanasia and worm counting, their worm burdens were compared with those from untreated control groups. At a dose rate of 1.25 mg/kg, monepantel showed efficacy above 91.9% against all major nematode species, with the exception of Chabertia ovina and Oesophagostomum venulosum. Efficacy against these two species was 93.6% and 94.0%, respectively, at a dose of 2.5 mg/kg. At this dose, efficacy was above 99.2% against nine other nematode species including Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp. and Nematodirus spp. It was concluded that 2.5 mg/kg would be a suitable dose rate for a commercial product.     

The microbial environment and intestinal nematode infections of Heligmosomoides polygyrus in laboratory mice

The difficulty of establishing primary infections of Heligmosomoides polygyrus (= Nematospiroides dubius) in ASH/CSI mice in the Laboratory Animal House at Royal Holloway and Bedford New College during a recent autumn and spring period was associated with a syndrome of worm distortion, together with zero or low worm establishment and reduced fecundity (eggs/female worm). The eggs produced were non-viable and the egg capsule comprised a rumpled lipid and ruptured chitin layer. The egg size and peaks of egg production were also reduced and the total egg output ceased entirely by day 28 post-infection in male mice. The syndrome was repeated when control LACA mice harbouring 'normal' infections of H. polygyrus were housed on the same source of peat bedding material as the ASH/CSI mice. An increase in H. polygyrus egg production in ASH/CSI mice, removed from the peat or treated with 0.04% oxytetracycline hydrochloride suggested that the cause of the syndrome was microbial in origin. A microbiological assay of the peat, which was the common denominator of all syndrome infections, revealed an abundance of chitinase secreting species of bacteria (Bacillaceae). Bacterial chitinase was therefore likely to rupture the chitin layer of the egg capsule producing nonviable eggs and either abnormal or no larvae. Preliminary in vitro studies using chitinase from Streptomyces griseus indicated that the hatching success of eggs of H. polygyrus was reduced as the concentration of chitinase increased.     

In vitro models of intestinal epithelial cell differentiation

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.     

In vitro efficacy of new antiprotozoals against Philasterides dicentrarchi (Ciliophora, Scuticociliatida)

Philasterides dicentrarchi is a histiophagous ciliate that causes severe losses in turbot and sea bass farming. This study investigated the in vitro efficacy against P. dicentrarchi of 85 newly synthesized compounds and 12 commercial compounds, of which 2 are fluoroquinolones (norfloxacine and lomefloxacine) with known antibacterial activity. Seventeen of the newly synthesized compounds (2 naphthyridines, 2 pyridothienodiazines and 13 pyridothienotriazines) and the fluoroquinolone norfloxacin showed good activity. The most promising compound was the pyridothienotriazine 12k, with activity similar to that of the salicylanilides niclosamide and oxiclozanide (MLC 0.8 mg l(-1) in PBS, 1.5 mg l(-1) in seawater; MLC = minimum 24 h lethal concentration).     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

In vitro and in silico antifungal efficacy of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani

We have investigated in vitro antifungal efficiency of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani (R. solani) plant pathogenic fungi. NCNH with size of 50–60 nm and concentrations of 10, 50, 100, and 150 μg mL^?1 were used. The results showed that growth of fungi in the presence of NCNH was significantly (p > .05) inhibited at 150 μg mL^?1 (85.13 ± .97) after 72 h. The results were validated through computational approaches. Molecular docking analysis of NCNH with endochitinase protein of R. solani was performed to validate the potential of antifungal activity of NCNH. Docking results showed different conformations of interaction of NCNH with endochitinase enzyme. The conformation with least binding energy ?13.54 kcal/mol was considered further. It is likely that NCNH interacts with the pathogens by mechanically wrapping, which may be one of the major toxicity actions of NCNH against R. solani. The analysis showed that NCNH might interwinds to endochitinase of R. solani leading to the deactivation of the enzyme. To best of our knowledge, this is the first report of antifungal efficacy of NCNH against R. solani and provides useful information about the application of NCNH in resisting crop disease.     

In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.     

In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.     

In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

In vitro and in vivo efficacy and in vitro metabolism of 1-phenyl-3-aryl-2-propen-1-ones against Plasmodium falciparum

Investigation of a series of 1-phenyl-3-aryl-2-propen-1-ones resulted in the identification of nine inhibitors with submicromolar efficacy against at least one Plasmodium falciparum strain in vitro. These inhibitors were inactive when given orally in a Plasmodium berghei infected mouse model. Significant compound degradation occurred upon their exposure to a liver microsome preparation, suggesting metabolic instability may be responsible for the lack of activity in vivo.     

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

In vitro and in vivo translational models for rare liver diseases

A challenge in developing effective treatments is the modeling of the human disease using in vitro and in vivo systems. Animal models have played a critical role in the understanding of disease pathophysiology, target validation, and evaluation of novel therapeutic agents. However, as the success rate from entry into clinical testing to drug approval remains low, it is critical to have high quality and well-validated models reflective of the disease condition. Additional experimental models are being developed based on functional in vitro 3D tissue models such as organoids and 3D bioprinted tissues. Because these 3D tissue models mimic closer the architecture, cell composition and physiology of native tissues, they are now being used as screening platforms in drug discovery and development and for tissue transplant in regenerative medicine. Here we review the current state-of-art of in vitro and in vivo translational models for the development of therapies for rare diseases of the liver.     

In vitro and in vivo activity of three sesquiterpenes against L(3) larvae of Anisakis type I

In order to investigate the possible use of terpenic derivatives to treat anisakiasis caused by L₃ larvae of Anisakis, we studied the in vitro and in vivo larvicidal activity of three sesquiterpenes (nerolidol, farnesol and elemol). In vitro experiments included the histological study of larval damage and in vivo studies the measurement of myeloperoxidase activity in rat gastrointestinal tract after administration of the sesquiterpenes. In the in vitro assays, the most active compound against the L₃ larvae was nerolidol, followed by farnesol; both caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture. In the in vivo assays, only 20% of infected rats treated with nerolidol or farnesol showed gastric wall lesions in comparison to 86.6% of control animals. According to these results, nerolidol and farnesol are good candidates for further research as biocidal agents against L₃ larvae of Anisakis type I.     

In vitro and in vivo investigations of human microsporidia.

The numerous infections of microsporidia which have been diagnosed in patients with AIDS have revealed the potential of these organisms for establishing themselves when the immune status of the host is compromised. Two species of Encephalitozoon, E. cuniculi and E. hellem, have been diagnosed in man, the former infecting a variety of tissues, the latter restricted to the corneal and conjunctival epithelia. These species are morphologically indistinguishable even at the ultrastructural level but can be separated biochemically. Two human sera were found to react with equal intensity in the ELISA on spores of E. cuniculi and E. hellem purified from in vitro cultures, and gave similar binding patterns in Western blots on SDS-PAGE protein profiles of the two species. This has raised questions about the identity of Encephalitozoon infections diagnosed previously in man. The diagnosis of Enterocytozoon bieneusi, which infects the intestinal enterocytes of AIDS patients and is associated with chronic diarrhoea, requires observation of smears or sections of biopsies or specialist observation of stool preparations. In vitro cultures, which would facilitate the raising of specific antisera, have proved difficult to establish. In vitro and in vivo systems for assaying drugs for microsporidia have revealed that albendazole has a marked effect on parasite numbers and morphology but does not eliminate infection, which resurges when drug pressure is removed.     

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.     

In vitro and in vivo action of recombinant human GM-CSF (rhGM-CSF) in patients with myelodysplastic syndromes

The availability of rhGM-CSF has allowed the in vivo treatment of patients with cytopenia. Therefore, a phase I-II trial was initiated to study the effect of rhGM-CSF in patients with myelodysplastic syndromes who were not eligible for other kinds of therapy. rhGM-CSF has been tested in 10 patients in doses from 15 micrograms/m2 to 150 micrograms/m2 given intravenously over 8 hours for a cycle of 7 days followed by an interval of 14 days and a second 7-day treatment course. A dose-dependent increase in leukocyte count was observed in 9 of 10 patients. No change in reticulocyte numbers was seen and only one patient experienced an increase in platelet count. Toxicity mainly consisted of mild phlebitis at the site of infusion and sternal pain after bolus injection. An increase in blast cell counts in some patients necessitated the start of low-dose Ara-C therapy.     

In vitro and in vivo interaction of synthetic peroxide RBx11160 (OZ277) with piperaquine in Plasmodium models

RBx11160 (OZ277) is a promising antimalarial drug candidate that Ranbaxy Laboratories Limited and Medicines for Malaria Venture (MMV) are currently developing as a fixed combination with piperaquine. Here, we describe the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activities of piperaquine in combination with RBx11160 and artemether. In vitro, both combinations demonstrated a slight tendency towards antagonism with mean sums of fractional inhibitory concentrations (mean Sigma FICs) of 1.5. In vivo, piperaquine and artemether were borderline antagonistic (mean Sigma FIC of 1.4). However, an additive in vivo interaction of piperaquine and RBx11160 (mean Sigma FIC of 1.1) was identified, suggesting that a RBx11160-piperaquine combination therapy in humans should allow each molecule to exert its full antimalarial effect.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.