Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome

Effect of chloroquine on gene expression of Plasmodium yoelii nigeriensis during its sporogonic development in the mosquito vector

<p>Abstract</p> <p>Background</p> <p>The anti-malarial chloroquine can modulate the outcome of infection during the <it>Plasmodium </it>sporogonic development, interfering with <it>Plasmodium </it>gene expression and subsequently, with transmission. The present study sets to identify <it>Plasmodium </it>genes that might be regulated by chloroquine in the mosquito vector.</p> <p>Methods</p> <p>Differential display RT-PCR (DDRT-PCR) was used to identify genes expressed during the sporogonic cycle that are regulated by exposure to chloroquine. <it>Anopheles stephensi </it>mosquitoes were fed on <it>Plasmodium yoelii nigeriensis</it>-infected mice. Three days post-infection, mosquitoes were fed a non-infectious blood meal from mice treated orally with 50 mg/kg chloroquine. Two differentially expressed <it>Plasmodium </it>transcripts (Pyn_chl091 and Pyn_chl055) were further characterized by DNA sequencing and real-time PCR analysis.</p> <p>Results</p> <p>Both transcripts were represented in <it>Plasmodium </it>EST databases, but displayed no homology with any known genes. Pyn_chl091 was upregulated by day 18 post infection when the mosquito had a second blood meal. However, when the effect of chloroquine on that transcript was investigated during the erythrocytic cycle, no significant differences were observed. Although slightly upregulated by chloroquine exposure the expression of Pyn_chl055 was more affected by development, increasing towards the end of the sporogonic cycle. Transcript abundance of Pyn_chl055 was reduced when erythrocytic stages were treated with chloroquine.</p> <p>Conclusion</p> <p>Chloroquine increased parasite load in mosquito salivary glands and interferes with the expression of at least two <it>Plasmodium </it>genes. The transcripts identified contain putative signal peptides and transmembrane domains suggesting that these proteins, due to their location, are targets of chloroquine (not as an antimalarial) probably through cell trafficking and recycling.</p

Antiplasmodial volatile extracts from Cleistopholis patens Engler & Diels and Uvariastrum pierreanum Engl. (Engl. & Diels) (Annonaceae) growing in Cameroon

In a search for alternative treatment for malaria, plant-derived essential oils extracted from the stem barks and leaves of Cleistopholis patens and Uvariastrum pierreanum (Annonaceae) were evaluated in vitro for antiplasmodial activity against the W2 strain of Plasmodium falciparum. The oils were obtained from 500 g each of stem barks and leaves, respectively, by hydrodistillation, using a Clevenger-type apparatus with the following yields: 0.23% and 0.19% for C. patens and 0.1% and 0.3% for U. pierreanum (w/w relative to dried material weight). Analysis of 10% (v/v) oil in hexane by gas chromatography and mass spectrometry identified only terpenoids in the oils, with over 81% sesquiterpene hydrocarbons in C. patens extracts and U. pierreanum stem bark oil, while the leaf oil from the latter species was found to contain a majority of monoterpenes. For C. patens, the major components were α-copaene, δ-cadinene, and germacrene D for the stem bark oil and β-caryophyllene, germacrene D, and germacrene B for the leaf oil. The stem bark oil of U. pierreanum was found to contain mainly β-bisabolene and α-bisabolol, while α- and β-pinenes were more abundant in the leaf extract. Concentrations of oils obtained by diluting 1-mg/mL stock solutions were tested against P. falciparum in culture. The oils were active, with IC50 values of 9.19 and 15.19 μg/mL for the stem bark and leaf oils, respectively, of C. patens and 6.08 and 13.96 μg/mL, respectively, for those from U. pierreanum. These results indicate that essential oils may offer a promising alternative for the development of new antimalarials

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseases

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.Portuguese Science and Technology Foundation (FCT) [PTDC/SAU-ORG/117266/2010, PTDC/BIM-MEC/1168/2012, UID/Multi/ 04326/2013]; FCT fellowships [SFRH/BPD/70277/2010, SFRH/BD/111824/2015