1,720,981 research outputs found
Mechanisms underlying Leishmania parasites survival and dissemination at late stage of infection in human macrophages
Get PDFLeishmania parasites are the causative agent of a wide range of human diseases known as leishmaniasis. The progression of disease pathogenesis is dependent on the ability of intracellular parasites to disseminate between human macrophages. It is assumed that parasites could exit macrophages following cell lysis. However, there is still a major gap in knowledge in understanding the exact mechanism pertaining to this critical step. This has been partially caused by the lack of reliable experimental models to study this stage of host-parasite interaction. This research aims to elucidate the mechanism behind cell-tocell spreading of Leishmania in vitro.
A link between host cell apoptosis and Leishmania propagation was initially established by determining the effect of apoptotic induction on infection over a period of 96 hours. The result showed that 60-90% of cells were inoculated with L. aethiopica and L. mexicana within 72 and 96 hours infection. Although, the concentration of viable cells was greatly reduced as compared to non-induced samples, a heavily infected cell population was obtained with this approach. Hence, this population of L. aethiopica and L. mexicana infected cells at 72 hours was used to infect differentiated THP-1 macrophages and validated as a model for infection spreading. Amastigotes spreading was detected within 12 hours of coculture as confirmed by flow cytometry and live cell imaging techniques. Interestingly, the live imaging revealed inter-cellular extrusion of L. aethiopica and L. mexicana from infected to a recipient cells without cell lysis. To address whether parasite extrusion is linked to apoptotic induction, this model was used to detect expression of several apoptotic markers. The results showed that L. aethiopica (but not L. mexicana) dissemination was correlated with host apoptotic induction, as there was a significant increase (p<0.05) in phosphatidyl serine (PS) externalization and active caspase-3 expression. In addition, western blot analysis demonstrated that L. aethiopica spread was associated with downregulation of three major apoptotic signaling pathways: AKT, NF-κB and PKC-δ. Further comparative proteomic analysis of this model supported these findings and showed that during L. aethiopica spreading, a number of host physiological processes were subverted. Specifically caspase-3 and caspase-9 activation strengthens the involvement of mitochondria in initiation of apoptosis. Interestingly, chitinase-3-like protein (inducer of IL-13 production) and butyrophilin (inhibitor of IFN-γ and IL-12) were markedly increased confirming the downregulation of inflammatory response. Finally, elevated increase in glutathione reductase and metallothionein-2 proteins, which are involved in scavenging free radicals, suggests that anti-microbial activity is also downregulated during L. aethiopica spreading.
In conclusion, by developing and validating for the first time a credible in vitro spreading model in human macrophages, this research has clarified the mechanism of Leishmania spreading. L. ethiopica promotes host cell apoptosis in order to disseminate from cell-to-cell without activating inflammatory and anti-microbial responses. In particular, apoptotic induction takes place via downregulation of AKT and NF-κB, release of cytochrome C and consequent activation of caspase-3 and PS exhibition, independently from PKC-δ. This model clearly fits with the silent spread of parasites that takes place in the first weeks (to months) of infection in mice model. Interestingly, the fact that L. mexicana utilizes a non-apoptotic strategy to spread suggests that alternative mechanisms of parasitic extrusion also exist underlying the necessity of species specific investigations. Understanding the molecular basis of Leishmania spreading contributes to filling a current gap in knowledge in the biology of Leishmania infection strategy of this very successful parasite. Moreover, it has the potential to open up a novel set of targets for drugs and vaccine development against this increasingly widespread neglected disease
Expression of green fluorescent protein (GFP) in Leishmania spp. to identify survival strategies the parasite imposes on the human host
Get PDFLeishmania protozoa are the etiological agent of human disease known as leishmaniasis. There are three main forms of this disease, whose manifestation depends on a number of factors including the infecting Leishmania species and the immunological status of the individual. Disease development requires extracellular form of the parasite, the promastigotes to be internalised, transform and replicate as intracellular amastigotes within mammalian cells. Once inside host cells parasites they regulate and control immune responses from inside the host cells through the modulation of protein expression including cytokines. The changes involving Leishmania promastigote and host cells is well documented during the early stage of infection. However, little is known on the modulative effect of L. aethiopica and L. mexicana during infection of host cells. It is key to have an altruistic approach to when studying the different Leishmania spp during host infection, as it is a key factor in the outcome of type of leishmaniasis.
The study of infection is complicated by the inherent difficulty of detecting intracellular parasites, as many methods are laborious and time consuming (microscopy with Giemsa staining). Implementation of fluorescent expressing parasites has been successfully used in a number of species to observe and quantify cellular infection. Although successful stable genetically modified Leishmania parasites are not available for L. mexicana and L. aethiopica, this research aims to investigate survival mechanisms such as cytokine and protein expression during the early stages of infection. This requires monitoring infection as the first step, which involved developing for the first time constitutively expressing GFP in L. aethiopica, L. major, L. mexicana and L. tropica. The effect of infection on host macrophages was directly quantified via comparative proteomics which identified significant differences in protein expression not only between uninfected and infected cells at late stages of infection but also between different species. The effect of infection on the ability of controlling host and neighbouring cells was further characterised by looking at cytokine & antimicrobial peptide expression, including TNFα, IL-10, TGFβ, IL-1RA and cathelicidin at various time points during L. aethiopica infection. As cytokine expression is closely linked to manipulation of T-cells, which are also know to be ultimately responsible for disease exacerbation/resolution, the effect of the parasites on such cells was further investigate. A novel role for T cells on infection was explored, specifically their ability to host parasites.
The successful stable integration of GFP expression in four Leishmania spp. was confirmed by PCR, western blot, fluorescent microscopy and flow cytometry (N=3). These parasites allowed the monitoring of infection in macrophages whilst investigating the expression of cytokines, proteins, and novel human cell types. Cathelicidin expression was shown to
increase during the early stages of infection at 8hrs following by a steady decline towards 48hrs post infection. The anti-inflammatory cytokine IL-10 expression was shown to increase from 2-48hrs peaking at the latest point with >3-fold difference post infection. Interestingly, the inflammatory cytokine TNFα showed expression increased from 2-48hrs, peaking with >5-fold at 48hrs post infection. Both these cytokines have been shown to peak at 8 hours followed by a decline, which may indicate subtle differences at a species (Lapara and Kelly 2010). Proteinomic study revealed L. mexicana and L. aethiopica both up-regulated a total of eight proteins with only two that were influenced by both species (N=3). In addition, down regulation was only identified during infection with L. aethiopica and not L. mexicana. However, during upregulation, the proteins identified were found to be involved in endocytosis (actin), immunological responses (MHC I), free radical scavenging and apoptosis (metallothionein). When comparing with similar proteomic studies by Singh et al, all modulation effects identified by L. aethiopica and L. mexicana were not previously shown during L. donovani infection. Infection was tested against human T lymphocytes as they have been implicated in the production of cytokines for the activation of macrophages allowing the elimination of intracellular parasites within macrophages. This novel work has shown L. aethiopica and L. mexicana are capable of infecting Jurkat cells (N=3) and enriched human T lymphocytes extracted from five different individuals. This finding has uncovered that infection is not only includes T cells (including CD4+ subsets) but percentage of infection is dependant on the species of parasites and the donor lymphocytes.
In conclusion, this research showed for the first time that L. aethiopica and L. mexicana modulate a repertoire of protein expression at 24hrs post infection. The changes of cytokines expression highlights differences between different species and the discovery that Leishmania has the ability to infect human T cells may commence the start of the complete understanding of the survival mechanisms imposed by the parasite on the wider cellular network. This will change the approach to studying immunology of leishmaniasis infection and potentially clarifying the role of these cells for which contrasting evidences in human and mice models have caused much debate
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Developing a single dressing containing both antifungal and antibacterial drugs for treating mixed infections in diabetic foot ulcers
Get PDFThe prevalence of chronic wounds such as diabetic foot ulcers (DFUs) is growing at an epidemic rate in the United Kingdom, with a high cost burden for the National Health Service and are prone to infections. Mixed bacterial-fungal infections are more complicated than single microbe infections with enhanced severity of DFUs. Moreover, recently developed medicated dressings target only either bacterial or fungal infection. This study therefore aims to develop advanced bioactive calcium alginate-based dressings that will deliver therapeutically relevant doses of broad-spectrum antibiotic and antifungal drugs to target mixed infections in DFUs. The therapeutic calcium alginate (CA) based dressings were prepared as films and wafers by solvent casting and freeze-drying technique respectively, with ciprofloxacin (CIP) and fluconazole (FLU) as model antibacterial and antifungal drugs respectively. The dressings were physically characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and texture analysis. Further functional characterization for exudate handling properties included porosity, water absorption (Aw), equilibrium water content (EWC), swelling index (Is), water vapour transmission rate (WVTR), evaporative water loss (EWL) and moisture content, drug dissolution by HPLC, in vitro antimicrobial and cell viability (biocompatibility) studies. Further, the exudate handling, antimicrobial and cell viability properties of the dressings were compared with commercial silver and CA based Algisite Ag® and hydrogel Actiformcool®. The drug loaded films were transparent, flexible, uniform, durable and elastic while drug loaded wafers were soft, uniform texture and thickness, and pliable in nature. SEM investigation showed differences in surface morphology between films (continuous sheets) and wafers (porous matrix). XRD confirmed the presence of calcium carbonate in the dressings and FTIR showed that the addition of drug caused the shift of peaks towards a higher wavenumber due to hydrogen bonding formation. Wafers showed better wound dressing properties than films and the commercial dressings for swelling, EWC (91 ̶ 95%), Aw (1085 ̶ 2377%) and moisture content (9 ̶ 17%) due to high porosity. The dressings showed initial fast release followed by sustained drug release which completely eradicated all causative bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and reduced fungal load (Candida albicans) 10 fold within 24 h. Moreover, the medicated dressings were found to be highly biocompatible (> 70% cell viability over 72 h) with human primary adult keratinocytes and showed maximum wound closure (in vitro scratch assay) within 7 days. CA wafers and films appeared to be potential delivery systems for delivering antimicrobial agents to mixed infected DFUs
Interaction between Leishmania parasites and mammalian macrophages
Get PDFLeishmania parasites are digenetic protozoans which infect human hosts and are causative agents of a series of diseases known under the name of leishmaniasis. Macrophages represent the main host. Hence the interaction between Leishmania and macrophages is a fundamental step in the development of the disease. Many studies have been undertaken to understand early stages of the parasite interaction with macrophages; however, few have investigated the later stages of infection. This study was undertaken to develop an experimental model to examine the fate of the parasites when they leave the safe environment represented by their host macrophage. Primarily, the study investigated how Leishmania spread to neighbouring cells without being recognized and killed by the immune system defences.
Three Old World species of Leishmania parasites: L. aethiopica, L. major and L. tropica, all responsible for the cutaneous form of the disease, were used. A model of infection was described using two cell lines well known for supporting infection: THP-1 and U937. Axenic amastogotes for L. aethiopica parasites were obtained and used to identify drugs active against the infection. On the basis of the information available in the literature, a model was suggested involving interaction of intracellular parasites with the host cells’ apoptotic machinery. Specifically it was suggested that Leishmania parasites were able to induce incomplete activation of apoptosis in the host cells. This hypothesis was confirmed by the findings that during infection an increased number of host cells showed two features associated with early apoptosis but not the one associated with the later stage. Results were validated in peripheral blood derived human macrophages. The information obtained from comparative proteomics analysis of the infection confirmed that Leishmania regulates apoptotic processes.
On the basis of the results obtained a model was presented to explain how induction of apoptosis allows intracellular amastigotes to spread unrecognised to uninfected macrophages without inducing an inflammatory response or losing the host cell’s protection
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
- …
