Infection of hematopoietic stem cells by Leishmania infantum increases erythropoiesis and alters the phenotypic and functional profiles of progeny

Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) – APQ-000647-13, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) by fellowship awarded to LRVA, OAMF and ATC. RCG received fellowship from CNPq program. DM is supported by a PhD fellowship SFRH/BD/91543/2012, MR is supported by a PhD fellowship SFRH/BD/89871/2012. RS is supported by FCT Investigator 2014 (IF/00021/2014)info:eu-repo/semantics/publishedVersio

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

ABSTRACT: Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. METHODS: The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using N(G)-methyl L-Arginine (N(G)MLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. RESULTS: INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with N(G)MLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. CONCLUSION: This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production

Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection

HIV/SIV Infection Primes Monocytes and Dendritic Cells for Apoptosis

Subversion or exacerbation of antigen-presenting cells (APC) death modulates host/pathogen equilibrium. We demonstrated during in vitro differentiation of monocyte-derived macrophages and monocyte-derived dendritic cells (DCs) that HIV sensitizes the cells to undergo apoptosis in response to TRAIL and FasL, respectively. In addition, we found that HIV-1 increased the levels of pro-apoptotic Bax and Bak molecules and decreased the levels of anti-apoptotic Mcl-1 and FLIP proteins. To assess the relevance of these observations in the context of an experimental model of HIV infection, we investigated the death of APC during pathogenic SIV-infection in rhesus macaques (RMs). We demonstrated increased apoptosis, during the acute phase, of both peripheral blood DCs and monocytes (CD14+) from SIV+RMs, associated with a dysregulation in the balance of pro- and anti-apoptotic molecules. Caspase-inhibitor and death receptors antagonists prevented apoptosis of APCs from SIV+RMs. Furthermore, increased levels of FasL in the sera of pathogenic SIV+RMs were detected, compared to non-pathogenic SIV infection of African green monkey. We suggest that inappropriate apoptosis of antigen-presenting cells may contribute to dysregulation of cellular immunity early in the process of HIV/SIV infection

Numerical and experimental study of diffraction by optical gratings in conical geometry at X-ray and UV wavelengths

L’utilisation de réseaux optiques dans la géométrie de diffraction conique a connu ces dernières décennies un essor remarquable dans les domaines UV et X grâce à ses propriétés particulières: absence de l’écrantage derrière les traits du réseau aux incidences rasantes, faible dispersion angulaire limitant l’étirement temporel, efficacité de diffraction élevée. Son usage s’est imposé pour la monochromatisation d’impulsions ultra-brèves. C’est aussi l’une des deux options retenues par la Nasa pour le spectrographe à réseau de l’Observatoire à rayon X de la future mission Lynx. Ce travail de thèse contribue au développement de méthodes numériques pour modéliser les effets de diffraction par des réseaux dans une géométrie encore peu étudiée sous cet aspect. La complexité de cette étude réside dans le couplage inhérent entre les deux états fondamentaux de polarisation. Du point de vue numérique, il impose un calcul ‘’vectoriel’’, là où, en géométrie classique des calculs scalaires suffisent. Notre travail s’est appuyé sur les méthodes numériques de calcul de diffraction par des structures périodiques déjà développées dans le cadre de la géométrie classique. Ces méthodes sont basées sur la théorie différentielle, qui consiste à propager une série d’ondes planes au travers de la zone modulée. La méthode différentielle employée est complétée par l’usage de l’algorithme de propagation de la matrice réflectivité. On contourne ainsi certains problèmes de convergence. Dans la partie théorique de ce travail, ces algorithmes sont étendus pour s’adapter aux cas de géométrie oblique. Sur cette base théorique, nous avons pu développer un code de calcul, nommé COROX, fonctionnant dans toutes les géométries d’utilisation. Un certain nombre de réseau types ont été étudiés, tant en géométrie oblique que classique, pour mettre en évidence, non seulement les efficacités de diffraction mais encore les effets de polarisation, (paramètres de Stokes et matrice de Müller) ainsi que les phases spectrales. Des propriétés intéressantes ont été remarquées, comme l’existence d’une composante circulaire non négligeable diffractée par réseau lamellaire quand l’onde incidente polarisée à 45° par rapport au plan du réseau. Le comportement de la phase spectrale est également une donnée significative pour une future gestion d’impulsions ultra-brèves. Des mesures de diffraction ont été effectuées sur la ligne Métrologie du Synchrotron SOLEIL, sur un réseau blazé de 150 traits/mm. Un accord raisonnable entre efficacités mesurées et calculées est constaté si l’on tient compte de la forte rugosité du réseau étudié.The conical geometry of optical grating diffraction has been suggested and studied, in the last 10 years, for cutting edge applications in the VUV and X-ray domains, due to its specific properties such as: absence of screen inside the grating grooves at grazing incidence, low angular dispersion which limits the temporal spread of short pulses, very high diffraction efficiencies. It has been accepted as the first choice technology for VUV short pulses monochromatization. It is also one of the two options selected by NASA, for the grating spectrograph of the future X-ray Observatory of the Lynx mission. This thesis reports our contribution to the development of numerical methods in order to model the effects of diffraction by optical gratings in this still little studied geometry. This study is made more complex by an inherent coupling between the two fundamental polarization modes. From the numerical aspect, it requires performing “vectorial” computations, whereas, in a classical diffraction geometry, scalar computations are sufficient. Our work is based on numerical methods already developed for modeling optical diffraction by periodic structures in the framework of classical geometry. These methods are using on the differential theory, whose main concept is propagating a set of plane waves throughout the modulated area. We use the differential method together with an algorithm of reflectivity matrix propagation. It overcomes some of the convergence issues. In the theoretical part of this work, reflectivity matrix algorithms are extended to the case of oblique geometry. On these theoretical grounds, we developed a computation code, named COROX, which can be applied in any geometry. A number of typical grating cases have been studied, both in the conical and of le classical one. The output is not only the diffraction efficiencies, but also the polarization properties (Stokes parameters, Müller matrix), as well as the spectral phases. Interesting properties have been noticed, such as the presence of a non-negligible circularly polarized component diffracted from a lamellar grating when the incident wave is linearly polarized at 45° from the grating plane. The spectral phase behavior is also a significant data for an eventual shape tayloring of ultrashort pulses. Diffraction efficiency measurements have been performed on the Metrology beamline of Synchrotron SOLEIL, using a 150 lines/mm blazed grating as a test object. A reasonable agreement between measured and computed efficiencies has been obtained, provided that the rather high roughness of this grating is taken into account

Étude numérique et expérimentale de la diffraction en géométrie conique de réseaux optiques aux longueurs d’ondes X et UV

The conical geometry of optical grating diffraction has been suggested and studied, in the last 10 years, for cutting edge applications in the VUV and X-ray domains, due to its specific properties such as: absence of screen inside the grating grooves at grazing incidence, low angular dispersion which limits the temporal spread of short pulses, very high diffraction efficiencies. It has been accepted as the first choice technology for VUV short pulses monochromatization. It is also one of the two options selected by NASA, for the grating spectrograph of the future X-ray Observatory of the Lynx mission. This thesis reports our contribution to the development of numerical methods in order to model the effects of diffraction by optical gratings in this still little studied geometry. This study is made more complex by an inherent coupling between the two fundamental polarization modes. From the numerical aspect, it requires performing “vectorial” computations, whereas, in a classical diffraction geometry, scalar computations are sufficient. Our work is based on numerical methods already developed for modeling optical diffraction by periodic structures in the framework of classical geometry. These methods are using on the differential theory, whose main concept is propagating a set of plane waves throughout the modulated area. We use the differential method together with an algorithm of reflectivity matrix propagation. It overcomes some of the convergence issues. In the theoretical part of this work, reflectivity matrix algorithms are extended to the case of oblique geometry. On these theoretical grounds, we developed a computation code, named COROX, which can be applied in any geometry. A number of typical grating cases have been studied, both in the conical and of le classical one. The output is not only the diffraction efficiencies, but also the polarization properties (Stokes parameters, Müller matrix), as well as the spectral phases. Interesting properties have been noticed, such as the presence of a non-negligible circularly polarized component diffracted from a lamellar grating when the incident wave is linearly polarized at 45° from the grating plane. The spectral phase behavior is also a significant data for an eventual shape tayloring of ultrashort pulses. Diffraction efficiency measurements have been performed on the Metrology beamline of Synchrotron SOLEIL, using a 150 lines/mm blazed grating as a test object. A reasonable agreement between measured and computed efficiencies has been obtained, provided that the rather high roughness of this grating is taken into account.L’utilisation de réseaux optiques dans la géométrie de diffraction conique a connu ces dernières décennies un essor remarquable dans les domaines UV et X grâce à ses propriétés particulières: absence de l’écrantage derrière les traits du réseau aux incidences rasantes, faible dispersion angulaire limitant l’étirement temporel, efficacité de diffraction élevée. Son usage s’est imposé pour la monochromatisation d’impulsions ultra-brèves. C’est aussi l’une des deux options retenues par la Nasa pour le spectrographe à réseau de l’Observatoire à rayon X de la future mission Lynx. Ce travail de thèse contribue au développement de méthodes numériques pour modéliser les effets de diffraction par des réseaux dans une géométrie encore peu étudiée sous cet aspect. La complexité de cette étude réside dans le couplage inhérent entre les deux états fondamentaux de polarisation. Du point de vue numérique, il impose un calcul ‘’vectoriel’’, là où, en géométrie classique des calculs scalaires suffisent. Notre travail s’est appuyé sur les méthodes numériques de calcul de diffraction par des structures périodiques déjà développées dans le cadre de la géométrie classique. Ces méthodes sont basées sur la théorie différentielle, qui consiste à propager une série d’ondes planes au travers de la zone modulée. La méthode différentielle employée est complétée par l’usage de l’algorithme de propagation de la matrice réflectivité. On contourne ainsi certains problèmes de convergence. Dans la partie théorique de ce travail, ces algorithmes sont étendus pour s’adapter aux cas de géométrie oblique. Sur cette base théorique, nous avons pu développer un code de calcul, nommé COROX, fonctionnant dans toutes les géométries d’utilisation. Un certain nombre de réseau types ont été étudiés, tant en géométrie oblique que classique, pour mettre en évidence, non seulement les efficacités de diffraction mais encore les effets de polarisation, (paramètres de Stokes et matrice de Müller) ainsi que les phases spectrales. Des propriétés intéressantes ont été remarquées, comme l’existence d’une composante circulaire non négligeable diffractée par réseau lamellaire quand l’onde incidente polarisée à 45° par rapport au plan du réseau. Le comportement de la phase spectrale est également une donnée significative pour une future gestion d’impulsions ultra-brèves. Des mesures de diffraction ont été effectuées sur la ligne Métrologie du Synchrotron SOLEIL, sur un réseau blazé de 150 traits/mm. Un accord raisonnable entre efficacités mesurées et calculées est constaté si l’on tient compte de la forte rugosité du réseau étudié

Inhibiting Human and Leishmania Arginases Using Cannabis sativa as a Potential Therapy for Cutaneous Leishmaniasis: A Molecular Docking Study

Cutaneous leishmaniasis (CL), a vector-borne parasitic disease caused by the Leishmania protozoan, is a serious public health problem in Morocco. The treatment of this disease is still based on pentavalent antimonials as the primary therapy, but these have associated side effects. Thus, the development of effective, risk-free alternative therapeutics based on natural compounds against leishmaniasis is urgent. Arginase, the key enzyme in the polyamine biosynthetic pathway, plays a critical role in leishmaniasis outcome and has emerged as a potential therapeutic target. The objective of this study was to test Cannabis sativa’s phytochemical components (cannabinoids and terpenoids) through molecular docking against Leishmania and human arginase enzymes. Our results showed that delta-9-tetrahydrocannabinol (THC) possessed the best binding energies of −6.02 and −6.35 kcal/mol with active sites of Leishmania and human arginases, respectively. Delta-9-THC interacted with Leishmania arginase through various amino acids including His139 and His 154 and linked to human arginase via His 126. In addition to delta-9-THC, caryophyllene oxide and cannabidiol (CBD) also showed a good inhibition of Leishmania and human arginases, respectively. Overall, the studied components were found to inhibit both arginases active sites via hydrogen bonds and hydrophobic interactions. These components may serve as therapeutic agents or in co-administrated therapy for leishmaniasis

Extraordinary transmission through subwavelength dielectric gratings in the microwave range

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