Leishmania amastigotes as targets for drug screening

Direct drug screening against the mammalian stage of Leishmania has been hampered by cost and the time consuming effort required to accomplish it. The ability to derive transgenic Leishmania expressing reporter genes opened up new possibilities for the development of drug screening tests. Further developments to standardize and gather multiple informations could now be envisionned. We will discuss on such available methodologies that could improve sensitivity, reliability, versatility and the rapidity, of the screen based on intracellular model

Experimental study of the function of the excreted/secreted Leishmania LmSIR2 protein by heterologous expression in eukaryotic cell line

BACKGROUND: In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells. RESULTS: Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection. CONCLUSIONS: LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed

A conceptual model for understanding the zoonotic cutaneous leishmaniasis transmission risk in the Moroccan pre-Saharan area

Publisher Copyright: © 2022 The AuthorsLeishmanioses are of public health concern in Morocco, mainly the Zoonotic Cutaneous Leishmaniasis (ZCL) endemic in the Moroccan pre-Saharian area. Transmission of this disease depends on eco-epidemiological and socio-economic conditions. Therefore, a multivariable approach is required to delineate the risk and intensity of transmission. This will help outline main disease risk factors and understand interactions between all underlying factors acting on disease transmission at a local and regional scale. In this context, we propose a new conceptual model, the Biophysical-Drivers-Response-Zoonotic Cutaneous Leishmaniasis (BDRZCL), adapted to the Pre-Saharian area. The proposed model highlights how the physical and human drivers affect the environment and human health. The incidence of ZCL is linked to human activity and biophysical changes or by their interactions. The human response added to risk drivers are the main components that influence the biophysical part. This model improves our understanding of the cause-effect interactions and helps decision-makers and stakeholders react appropriately.publishersversionpublishe

LEISHMANIA SPF?: COMPLETELY DEFINED MEDIUM WITHOUT SERUM AND MACROMOLECULES (CDM/LP) FOR THE CONTINUOUS ZN VZTRO CULTIVATION OF INFECTIVE PROMASTIGOTE FORMS

Abstract. The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RE" 1640 medium for all Leish?nania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications. Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical areas, which include, cutaneous, mucocutaneous, and visceral leishmaniasis. The parasite undergoes a digenic life cycle between a nonmotile intracellular amastigote stage parasitizing the mammalian phagocytic cells and a flagellated, motile promastigote stage in the midgut of its sandfly vector.' A similar promastigote form develops when parasites are cultured in cell-free medium.2 In vitro standardized cultivation of the members of the Leishmania genus is a useful approach for yielding amount of parasites suitable for diagnosis purposes to provide a better knowledge of host-parasite relationships and for the determination of biologic and immunologic characteristics of the parasite. One of the primary goals of culturists has been to achieve the long-term maintenance of active and dividing populations of different Leishmania species. The different media developed over the past 90 years can be classified in two major categories: semi-solid biphasic media and liquid monophasic media. Leishmania promastigotes were first grown on diphasic blood agar (NNN),3.4 which was later enriched with bacteriologic additives such as brain heart infusion: and is used today with various modifications of the liquid phase added to the solid These nondefined diphasic media are still used today for adaptation and cultivation of Leishmania strains directly isolated from both vertebrate and invertebrate hosts. However, they have some disadvantages. For example, they are complex to prepare and difficult to standardize. Moreover, they contained blood and bacteriologic additives as important factors for parasite replication, which complicate biologic and immunologic studies. Furthe; progress has been made with the use of serumenriched liquid monophasic media. Nondefined glucose-lactalbumin-serum-hemoglobin medium, originally developed for African trypanosomes,lo.ll liyer infusion tryptose (LIT) medium,12 Panmede medium,13 modified NIH medium using I ---hemolyzed rabbit blood,14 and semi-defined HOMEN's medium15 have been reported. Serum-emiched tissue culture media, initially formulated for the cultivation of mammalian or insect cells, have been successfully used to cultivate several Old and New World Leishmania species. These include 199H medium, RPMI 1640 medium, Dulbecco's minimal essential medium,5.16 Schneider's Drosophila medium, and Grace's insect tissue culture medium.16-18 However, all of these tissue culture media require addition of high concentrations of fetal bovine serum (10-30%) as an essential factor for long-term growth of Leishmania promastigotes. Defined media were developed for the cultivation of certain Leishmania. Leishmania tarentolae has been cultured in a chemically defined medium;lg however, this medium could not support the growth of other Leishmania species. Attempts to replace serum by bovine serum albuminzo.21 or mixture of purine bases, vitamins, ;utd bovine albumin fraction IVz2 were also made for cultivating L. donovani promastigote forms. An easily prepared, nearly defìned medium containing salts, glucose, and tryptose (i.e., an LIT medium), to which was added concentrated RPMI 1640 and 199 media, was shown to be adequate for the cultivation of L. chagasi and L. amazonensis proma~tigotes~~ and other Leishmania species.z4 Media developed for serum-free growth of mammalian cells were adapted for the serial cultivation of different Leishmania species. These media were enriched with large concentrations of certain amino acids, vitamins, bovine albumin, hormones, peptide supplement~,2~-~~ or more recently, with human urine.32.33 In the present paper, we report a completely chemically defined culture medium free of serum, macromolecules, proteins, and peptides that readily supports the growth and maintenance of promastigote forms of various Leishmania species without compromising parasite growth rates. Morphologic, biochemical, immunologic, and biologic properties of asencally grown promastigote forms are presented. 41 Fonds Docu meritai re O RSTGP

Training Children to Perceive Non-native Lexical Tones: Tone Language Background, Bilingualism, and Auditory-Visual Information

This study investigates the role of language background and bilingual status in the perception of foreign lexical tones. Eight groups of participants, consisting of children of 6 and 8 years from one of four language background (tone or non-tone) × bilingual status (monolingual or bilingual)—Thai monolingual, English monolingual, English-Thai bilingual, and English-Arabic bilingual were trained to perceive the four Mandarin lexical tones. Half the children in each of these eight groups were given auditory-only (AO) training and half auditory-visual (AV) training. In each group Mandarin tone identification was tested before and after (pre- and post-) training with both auditory-only test (ao-test) and auditory-visual test (av test). The effect of training on Mandarin tone identification was minimal for 6-year-olds. On the other hand, 8-year-olds, particularly those with tone language experience showed greater pre- to post-training improvement, and this was best indexed by ao-test trials. Bilingual vs. monolingual background did not facilitate overall improvement due to training, but it did modulate the efficacy of the Training mode: for bilinguals both AO and AV training, and especially AO, resulted in performance gain; but for monolinguals training was most effective with AV stimuli. Again this effect was best indexed by ao-test trials. These results suggest that tone language experience, be it monolingual or bilingual, is a strong predictor of learning unfamiliar tones; that monolinguals learn best from AV training trials and bilinguals from AO training trials; and that there is no metalinguistic advantage due to bilingualism in learning to perceive lexical tones

A Histone Deacetylase (HDAC) inhibitor with pleiotropic in vitro anti-toxoplasma and anti-plasmodium activities controls acute and chronic toxoplasma infection in mice

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound's efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence-structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.This research was funded by IDEX Innovation Grant, UGA, 2017 and The GIS ChemBioFranceinfo:eu-repo/semantics/publishedVersio

Development of a murine infection model with Leishmania killicki, responsible for cutaneous Leishmaniosis in Algeria : application in pharmacology

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime (R) significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria

Descriptive and Geometric Morphometry of the Wings of Phlebotomus sergenti Populations in Central Morocco

Background: Phlebotomus sergenti, the proven vector of Leishmania tropica, the causative agent of anthroponotic cu­taneous leishmaniasis, is widely distributed in Morocco. Previous works using molecular markers (Internal Transcribed Spacer 2 rDNA and Cytochrome B mtDNA) hypothesized the existence of multiple closely related populations of sand fly species (cryptic species) that would exhibit distinct vectorial capacities. This work studies morphotypic diversity using traditional and geometric morphometry analyses carried out on Ph. sergenti's wings from central Morocco, where active L. tropica transmission occurs for 30 years. Methods: Descriptive characteristics (size and shape) of the right wings were measured in Ph. sergenti’s specimens collected from fourteen stations in central Morocco. Both traditional and geometric morphometry methods were used to analyse geographic variations in Ph. sergenti wing’s size and shape. Results: These analyses support the existence of distinct Ph. sergenti populations, enlightening significant phenotypic variations of Ph. sergenti’s wings, regarding their size and shape, depending on geographic origin. In addition, tradi­tional and geometric morphometric analyses of the wing’s length, centroid size, β, ɵ, and γ distances allowed clear dis­crimination of Ph. sergenti sub-populations. Conclusion: These data pinpoint the adaptative ability of Ph. sergenti to local environmental conditions. Additional studies are now required to further shed light on the genetic structure of Ph. sergenti populations in Morocco

Induction of a Peptide with Activity against a Broad Spectrum of Pathogens in the Aedes aegypti Salivary Gland, following Infection with Dengue Virus

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide

Exploring the mass and redshift dependence of the cluster pressure profile with stacks on thermal SZ maps

We provide novel constraints on the parameters defining the universal pressure profile (UPP) within clusters of galaxies, and explore their dependence on the cluster mass and redshift, from measurements of Sunyaev-Zel'dovich Compton-$y$ profiles. We employ both the $\textit{Planck}$ 2015 MILCA and the ACT-DR4 $y$ maps over the common $\sim 2,100\,\text{deg}^2$ footprint. We combine existing cluster catalogs based on KiDS, SDSS and DESI observations, for a total of 23,820 clusters spanning the mass range $10^{14.0}\,\text{M}_{\odot}<M_{500}<10^{15.1}\,\text{M}_{\odot}$ and the redshift range $0.02<z<0.98$. We split the clusters into three independent bins in mass and redshift; for each combination we detect the stacked SZ cluster signal and extract the mean $y$ angular profile. The latter is predicted theoretically adopting a halo model framework, and MCMCs are employed to estimate the UPP parameters, the hydrostatic mass bias $b_{\rm h}$ and possible cluster miscentering effects. We constrain $[P_0,c_{500},\alpha,\beta]$ to $[5.9,2.0,1.8,4.9]$ with $\textit{Planck}$ and to $[3.8,1.3,1.0,4.4]$ with ACT using the full cluster sample, in agreement with previous findings. We do not find any compelling evidence for a residual mass or redshift dependence, thus expanding the validity of the cluster pressure profile over much larger $M_{500}$ and $z$ ranges; this is the first time the model has been tested on such a large (complete and representative) cluster sample. Finally, we obtain loose constraints on the hydrostatic mass bias in the range 0.2-0.3, again in broad agreement with previous works.Comment: 39 pages, 22 figures. Accepted for publication in Apj