Some factors affecting the cultivationin vitroof the erythrocytic stages ofPlasmodium knowlesi

Parasitology ◽  
1969 ◽  
Vol 59 (4) ◽  
pp. 915-924 ◽  
Author(s):  
P. I. Trigg
Keyword(s):  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Host Cells ◽  
World Health ◽  
Vitamin B ◽  
Low Oxygen ◽  
New Host ◽  
Factors Affecting ◽  
Oxygen Tensions

A study was made of some of the factors which affect the growth ofPlasmodium knowlesiin blood diluted in the Harvard medium. Some slight enhancement of growth and re-invasion of new host cells was obtained whenP. knowlesiwas incubated in low oxygen tensions. Some growth but no re-invasion was obtained under anaerobic conditions. High oxygen tensions were also inhibitory.The lipids present in the plasma are important for the growth ofP. knowlesi in vitro.Stearic acid and cholesterol are required by the parasite but do not account for all the growth promoting properties of plasma. A requirement for vitamin B12was also demonstrated.I should like to thank Dr F. Hawking and Dr J. Williamson for their advice and for reading through the manuscript, Mr T. J. Scott-Finnigan for technical assistance, Dr K. Mervyn (Glaxo Research Ltd.) for samples of vitamin B12analogues and the Hachmeister Company (Pittsburgh) for a sample of TEM 4T. This work received financial assistance from the World Health Organization.

The use of proprietary tissue-culture media for the cultivation in vitro of the erythrocytic stages of Plasmodium knowlesi

Parasitology ◽  
1969 ◽  
Vol 59 (4) ◽  
pp. 925-935 ◽  
Author(s):  
P. I. Trigg
Keyword(s):  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Culture Media ◽  
Host Cells ◽  
World Health ◽  
New Host ◽  
Stage Of Development ◽  
Perfusion Apparatus ◽  
Cultivation In Vitro

An improved perfusion apparatus for the cultivation of malaria parasites is described. It consists of a sandwich of three chambers, of which the outer two contained medium and the inner contained the infected blood sample.Using this apparatus and rocker-dilution cultures it has been shown that medium ‘199’ and medium ‘NCTC 135’ are as good as the Harvard medium for the cultivation of P. knowlesi in vitro.The asexual cycle of P. knowlesi took slightly longer in vitro than in vivo. The stage of development at which the parasite was inoculated into culture affected the amount of reinvasion of new host cells. Thus, a greater amount of re-invasion was obtained with an inoculum of schizonts than with one of ring stage parasites. The rate of multiplication of the parasite decreased at each successive subculture.I should like to thank Dr F. Hawking and Dr J. Williamson for their advice, Mr T. J. Scott-Finnigan for technical assistance, Mr F. R. Wanless and Mr C. D. Sutton for taking the photographs, and Mr F. A. New for drawing the figures. The perfusion apparatus was made by Mr T. Harding of the Engineering Division, National Institute for Medical Research. This work received financial assistance from the World Health Organization.

Short-term culture ofPlasmodium knowlesi

Parasitology ◽  
1971 ◽  
Vol 62 (2) ◽  
pp. 309-320 ◽  
Author(s):  
G. A. Butcher ◽  
S. Cohen
Keyword(s):  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Culture Method ◽  
World Health ◽  
Leucine Incorporation ◽  
Parasite Protein ◽  
Average Multiplication ◽  
Parasite Multiplication ◽  
Health Organization

The culture method developed by Geiman and co-workers has been modified to give average multiplication rates forPlasmodium knowlesiof at least six-fold in 24 h.Individual modifications have been assessed on the basis of parasite multiplication and [3H]leucine incorporation into parasite protein. The second cycle ofin vitrodevelopment which is particularly influenced by the conditions of culture was improved by: (i) culture of washed parasitized red cells in a medium containing dialysed homologous serum screened for its ability to support parasite growth; (ii) addition of ATP, Co-enzyme A (or pantothenate) and glutamine.We thank Mr E. D. Dennis and Miss Susan Wanstall for technical assistance. This work was supported by grants from the Medical Research Council and the World Health Organization.

Action of pyrimethamine and related drugs againstPlasmodium knowlesi in vitro

Parasitology ◽  
1971 ◽  
Vol 62 (3) ◽  
pp. 431-444 ◽  
Author(s):  
W. E. Gutteridge ◽  
P. I. Trigg
Keyword(s):  
Dihydrofolate Reductase ◽  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Protein Hydrolysate ◽  
Immune Serum ◽  
World Health ◽  
Malarial Parasite ◽  
Schizont Stage ◽  
Free System

A dihydrofolate reductase has been isolated from free-parasite preparations of the primate malarial parasite,Plasmodium knowlesi. Its properties are similar to those reported for the enzyme from the rodent malarial parasite,P. berghei,even though the base compositions of the DNA of these two species are quite different. TheKmsfor substrate and cofactor are 3 × 10−6Mand 1 × 10−6Mrespectively and the molecular weight is ˜ 200000. Concentrations of pyrimethamine and trimethoprim as low as 1 × 10−9Mand 3 × 10−8Mrespectively are sufficient to cause 50% inhibition of enzyme activity. The sensitivity to inhibition by pyrimethamine and trimethoprim of growth of cultures ofP. knowlesihas also been investigated. Preliminary experiments showed that it was only the schizont stage that was susceptible to the action of the drugs and that in their presence, normal nuclear divisions and segmentation did not occur and subsequently, no reinvasion of fresh red cells took place. The minimum concentrations of drug required to produce these effects were 10−9Mfor pyrimethamine and 10−7Mfor trimethoprim. Thus, there is a close correlation between the concentrations of pyrimethamine and trimethoprim required to inhibit the dihydrofolate reductase in a cell-free system and the growth of the parasitein vitro. Pyrimethamine (10−9M) did not, however, affect the incorporation of radioactive precursors into DNA, RNA or protein of schizont stage parasites until after morphological damage could be seen and reinvasion was complete in control cultures. The time courses of incorporation of [14C]algal protein hydrolysate into protein in the presence (10−9M) or absence of pyrimethamine are the same as those described recently with immune serum. The possibility is thus raised as to whether pyrimethamine and immune serum act in the same way.One of us (P.I.T.) received financial assistance from the World Health Organization. We thank Dr F. Hawking for many helpful discussions, Miss Jane Dunnett and Mr T. Scott-Finnigan for technical assistance and Dr O. D. Standen for samples of pyrimethamine and trimethoprim.

Effects of chloroquine on Plasmodium knowlesi in vitro

Parasitology ◽  
1972 ◽  
Vol 64 (1) ◽  
pp. 37-45 ◽  
Author(s):  
W. E. Gutteridge ◽  
P. I. Trigg ◽  
P. M. Bayley
Keyword(s):  
Mammalian Cells ◽  
Thermal Denaturation ◽  
Technical Assistance ◽  
Plasmodium Knowlesi ◽  
Fluorescence Emission ◽  
World Health ◽  
Isosbestic Point ◽  
Dna And Rna ◽  
Health Organization

The binding of chloroquine to DNA isolated from P. knowlesi has been investigated. In the presence of increasing concentrations of malarial DNA, the absorption spectrum of chloroquine exhibited strong hypochromism in the range 325–350 nm, small red shifts of the absorption maxima and an isosbestic point at 300 nm. The fluorescence emission of the drug at 380 nm (excitation 330 nm) was quenched five-fold by malarial DNA. Chloroquine itself protected malarial DNA from thermal denaturation. These data are consistent with binding of the drug to malarial DNA as has been suggested from studies with DNA from bacteria and mammalian cells. The binding affinity for malarial DNA is of the same order as that for mammalian DNA.The effect of chloroquine on the metabolism of P. knowlesi in culture was also investigated. There was a direct correlation between concentrations of drug which affect the morphology of parasites and those which affect markedly the incorporation of 3H-adenosine into DNA and RNA, 14C-isoleucine incorporation into protein and the formation of lactate as a result of respiration. However, all processes were inhibited to the same extent, indicating that binding to DNA is not the only action of the drug.The mode of action of chloroquine and the question of resistance to it are discussed.One of us (P. I. T.) received financial assistance from the World Health Organization. We thank Dr F. Hawking and Dr J. Williamson for many helpful discussions, Dr K. N. Brown for reading through the manuscript, and Miss Jane Dunnett, Mrs A. C. Gutteridge and Mr T. Scott-Finnigan for skilled technical assistance.

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity

2021 ◽  
Author(s):  
Daniel Morgan Foulkes ◽  
Keri McLean ◽  
Marta Sloniecka ◽  
Dominic Byrne ◽  
Atikah S Haneef ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Opportunistic Pathogen ◽  
World Health Organisation ◽  
Host Cells ◽  
World Health ◽  
Corneal Epithelial Cell ◽  
Cell Infection ◽  
Minimal Inhibitory Concentrations ◽  
Line Of Therapy

Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.

scholarly journals Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fernando J Sánchez-Valdéz ◽  
Angel Padilla ◽  
Wei Wang ◽  
Dylan Orr ◽  
Rick L Tarleton
Keyword(s):  
Trypanosoma Cruzi ◽  
Drug Treatment ◽  
Drug Exposure ◽  
Host Cells ◽  
New Host ◽  
Cytotoxic Compounds ◽  
Drug Treatments ◽  
Disease Agent

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

Production of in vitro bovine embryos supplemented with l-carnitine in different oxygen tensions and the relation to nitric oxide

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 403-408
Author(s):  
Daniela Moraes Pereira ◽  
Christopher Junior Tavares Cardoso ◽  
Wilian Aparecido Leite da Silva ◽  
Mirela Brochado Souza-Cáceres ◽  
Mariana Santos ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
Calf Serum ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Cleavage Rate ◽  
Treatment Groups ◽  
Oxygen Tensions

SummaryThe aim of this study was to evaluate the production of bovine embryos in vitro when supplemented with l-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) in in vitro culture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC; n = 837) were matured in vitro for 24 h and fertilization was performed for 18 h. Zygotes were cultured in vitro for 9 days after in vitro fertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2 tension either with or without the addition of 3.03 mM l-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P > 0.05) between high and low O2 tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P < 0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2 tension (P < 0.1). The addition of 3.03 mM l-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension without l-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2 tension.

First Example of Antiparasitic Activity Influenced by Thermochromism: Leishmanicidal Evaluation of 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine Metal Complexes

2020 ◽  
Vol 16 (3) ◽  
pp. 422-430 ◽  
Author(s):  
José M. Méndez-Arriaga ◽  
Itziar Oyarzabal ◽  
Álvaro Martín-Montes ◽  
Judith García-Rodríguez ◽  
Miguel Quirós ◽  
...  
Keyword(s):  
Physical Properties ◽  
Metal Complexes ◽  
New Drugs ◽  
Host Cells ◽  
World Health ◽  
Neglected Diseases ◽  
Leishmania Spp ◽  
Different Strains ◽  
Health Organization

Background: The World Health Organization catalogues illnesses such as Leishmaniasis as neglected diseases, due to low investment in new drugs to fight them. The search of novel and non-side effects anti-parasitic compounds is one of the urgent needs for the Third World. The use of triazolopyrimidines and their metallic complexes has demonstrated hopeful results in this field. Objective: This work studies the antiparasitic efficacy of a series of 5,7-dimethyl-1,2,4- triazolo[1,5-a]pyrimidine first row transition metal complexes against three leishmania spp. strains. Methods: The in vitro antiproliferation of promastigote forms of different strains of leishmania spp. (L. infantum, L. braziliensis and L donovani) and the cytotoxicity in macrophage host cells are reported here. The antiparasitic assays have been complemented with enzymatic tests to elucidate the mechanisms of action. New crystal structure description, thermal analysis, magnetic susceptibility and magnetization experiments have also been carried out in order to present a whole characterization of the studied compounds and interesting physical properties besides the biological tests. Results: The results of antiproliferation screening and cytotoxicity show great antiparasitic efficacy in the studied complexes. The superoxide dismutase enzymatic assays exhibit a different behaviour according to the thermochromic triazolopyrimidine form tested. Conclusion: Antiproliferative assays and enzymatic tests corroborate the synergetic leishmanicidal effect present in coordination triazolopyrimidine complexes. The changes in coordination sphere derived from thermochromism affect the physical properties as well as the biological efficacy.

scholarly journals A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U

2021 ◽  
Vol 478 (3) ◽  
pp. 647-668
Author(s):  
Daniel M. Foulkes ◽  
Keri McLean ◽  
Yalin Zheng ◽  
Joscelyn Sarsby ◽  
Atikah S. Haneef ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
T Cells ◽  
Bacterial Load ◽  
World Health Organisation ◽  
Host Cells ◽  
World Health ◽  
Infection Model ◽  
Compound A ◽  
Type 3

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

scholarly journals Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

2017 ◽  
Author(s):  
Fernando J. Sánchez-Valdéz ◽  
Angel Padilla ◽  
Wei Wang ◽  
Dylan Orr ◽  
Rick Tarleton
Keyword(s):  
Trypanosoma Cruzi ◽  
Drug Treatment ◽  
Drug Exposure ◽  
Host Cells ◽  
New Host ◽  
Cytotoxic Compounds ◽  
Drug Treatments ◽  
Disease Agent

AbstractThe ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy pathway in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

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