Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Over its life cycle, the <it>Plasmodium falciparum </it>parasite is exposed to different environmental conditions, particularly to variations in O₂pressure. For example, the parasite circulates in human venous blood at 5% O₂pressure and in arterial blood, particularly in the lungs, at 13% O₂pressure. Moreover, the parasite is exposed to 21% O₂levels in the salivary glands of mosquitoes.</p> <p>Methods</p> <p>To study the metabolic adaptation of <it>P. falciparum </it>to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken.</p> <p>Results</p> <p>Even though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response.</p> <p>Conclusions</p> <p>These results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of <it>P. falciparum</it>. This study provides a better understanding of the adaptive capabilities of <it>P. falciparum </it>to environmental changes and may lead to the development of novel therapeutic targets.</p

Hétérogénéité phénotypique et fonctionnelle des lymphocytes T CD4+ intrafolliculaires dans le lymphome folliculaire

Le lymphome folliculaire (FL) est le lymphome non Hodgkinien indolent le plus fréquent. Malgré une stabilisation de l'incidence et une diminution de la mortalité dues aux nouvelles stratégies thérapeutiques, la médiane de survie pour les patients souffrant de cette tumeur reste d'environ 10 ans. Cette pathologie se caractérise par une accumulation de lymphocytes B tumoraux matures dans les ganglions. Le microenvironnement non tumoral a un rôle prépondérant dans le maintien et la survie des cellules tumorales. Parmi les compartiments composant celui-ci, nous nous sommes plus particulièrement intéressés aux T CD4+ intrafolliculaires. Nous avons ainsi mis en évidence la présence de T follicular helper (TFH) soutenant la croissance des cellules B tumorales. Bien que ces TFH ont un profil d'expression génique proche de celui des TFH d'amygdales, certaines disparités existent entre ces 2 populations. Entre autre, nous constatons une expression plus importante de CD40L et d'IL4 dans les TFH de FL, molécules toutes deux impliquées dans la survie des B tumoraux face à la mort induite par le complément Rituximab-dépendante. Nous montrons également la présence d'une population de Treg intrafolliculaires, que nous avons appelé T régulateurs folliculaires (TFR). Bien que partageant des caractéristiques phénotypiques proches des TFH, ces TFR possèdent uniquement une activité suppressive face à des lymphocytes T conventionnels Ainsi nous décrivons dans le FL, deux populations partageant des caractéristiques phénotypiques, mais qui fonctionnellement agissent différemment: soit directement sur la cellule tumorale en permettant sa survie, soit en inhibant les cellules T effectrices.Follicular lymphoma (FL) is the most common indolent non Hodgkin s lymphoma. Despite a stabilization of incidence and a decreased mortality owing to new drug strategies, survival median is about only 10 years. This tumor is characterized by an accumulation of tumor B cells in lymph nodes. Importantly, the non-tumor microenvironment has a pivotal role in the maintenance and survival of FL B cells. Among the microenvironment-forming-cells, we focused on intrafollicular CD4+ T cells. We showed that intrafollicular CD4+ T cells comprised follicular helper T cells (TFH) supporting the tumor B cell growth. Despite the similarities between FL and tonsil TFH gene expression profile, we revealed some discrepancies, and showed that FL TFH expressed more CD40-L and IL-4 than tonsil TFH. Interestingly, these two factors were able to rescue FL tumor B cells from Rituximab mediated complement-dependent cell apoptosis. In addition, we also revealed the presence of intrafollicular Treg, called TFR. Despite their similar phenotype with TFH, TFR exerted a strong suppressive activity against effector T cells. Overall, we described in the FL microenvironment the presence of two CD4+ T cell subsets sharing similar membrane phenotype but displaying opposite functional properties: either a direct growth effect on tumor FL B cells, or a suppressive activity on effector T cells.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Evaluation of whole-blood conservation reagents for Hematoflow-based WBC differential count: Unsatisfactory results

International audienceBackground:As conservation of whole blood samples undergoing white blood cell (WBC) differential performed by flow cytometry (Hematoflow) is needed, we evaluated the effects of two commercially available fixatives, namely TransfixTM and Streck Cell PreservativeTM.Methods:We focused on 15 normal samples and on 13 various pathological samples. We compared the two fixatives and cold- or room- temperature effects on various parameters provided by the Hematoflow system.Results:We observed that, even after 2 hours of sample treatment, the conservative methods led to significant modifications of the cell percentages due to substantial variations of the epitope expression.Conclusion:None of the different conservation methods is really reliable for WBC differential performed by flow cytometry and thus samples should be analyzed promptly or stored at 4°C

Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and CD40L signaling.

International audienceInterleukin-15 (IL-15) has been extensively studied for its role in the survival and proliferation of NK and T cells through a unique mechanism of trans-presentation by producer cells. Conversely, whereas activated B cells have been described as IL-15-responding cells, the cellular and molecular context sustaining this effect remains unexplored. In this study, we found that, whereas human B cells could not respond to soluble IL-15, monocytes and lymphoid tissue-derived macrophages but not stromal cells efficiently trans-present IL-15 to normal B cells and cooperate with T-cell-derived CD40L to promote IL-15-dependent B-cell proliferation. Furthermore, CD40L signaling triggers a Src-independent upregulation of STAT5 expression and favors a Src-dependent phosphorylation of STAT5 in response to IL-15. In follicular lymphoma (FL), immunohistochemical studies reported a strong relationship between malignant B cells, infiltrating macrophages and T cells. We show here an overexpression of IL-15 in purified tumor-associated macrophages, and STAT5A in purified tumor B cells. Moreover, FL B cells respond to IL-15 trans-presented by monocytes/macrophages, in particular, in the presence of CD40L-mediated signaling. This cooperation between IL-15 and CD40L reinforces the importance of tumor microenvironment and unravels a mechanism of FL growth that should be considered if using IL-15 as a drug in this disease

CD10 delineates a subset of human IL-4 producing follicular helper T cells involved in the survival of follicular lymphoma B cells.

International audienceIn follicular lymphoma (FL), follicular helper T cells (TFH) have been depicted as one of the main components of the malignant B-cell niche and a promising therapeutic target. Although defined by their capacity to sustain FL B-cell growth together with specific gene expression and cytokine secretion profiles, FL-TFH constitute a heterogeneous cell population. However, specific markers reflecting such functional heterogeneity are still lacking. In this study, we demonstrate that CD10 identifies a subset of fully functional germinal center TFH in normal secondary lymphoid organs. Importantly, this subset is amplified in the FL context, unlike in other B-cell lymphomas with a follicular growth pattern. Furthermore, whereas FL-TFH produce high levels of interleukin (IL)-21 and low levels of IL-17 irrespectively of their CD10 expression, CD10pos FL-TFH specifically exhibit an IL-4hiIFN-γloTNF-αhi cytokine profile associated with a high capacity to sustain directly and indirectly malignant B-cell survival. Altogether, our results highlight the important role of this novel functional subset in the FL cell niche

Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.: Intratumoral TFH in follicular lymphoma

International audienceAccumulating evidences indicate that the cellular and molecular microenvironment of follicular lymphoma (FL) has a key role in both lymphomagenesis and patient outcome. Malignant FL B cells are found admixed to specific stromal and immune cell subsets, in particular CD4(pos) T cells displaying phenotypic features of follicular helper T cells (T(FH)). The goal of our study was to functionally characterize intratumoral CD4(pos) T cells. We showed that CXCR5(hi)ICOS(hi)CD4(pos) T cells sorted from FL biopsies comprise at least two separate cell populations with distinct genetic and functional features: (i) CD25(pos) follicular regulatory T cells (T(FR)), and (ii) CD25(neg) T(FH) displaying a FL-B cell supportive activity without regulatory functions. Furthermore, despite their strong similarities with tonsil-derived T(FH), purified FL-derived T(FH) displayed a specific gene expression profile including an overexpression of several genes potentially involved directly or indirectly in lymphomagenesis, in particular TNF, LTA, IL4 or CD40LG. Interestingly, we further demonstrated that these two last signals efficiently rescued malignant B cells from spontaneous and rituximab-induced apoptosis. Altogether, our study demonstrates that tumor-infiltrating CD4(pos) T cells are more heterogeneous than previously presumed, and underlines for the first time the crucial role of T(FH) in the complex set of cellular interactions within FL microenvironment

Tacrolimus diffusion across the peripheral mononuclear blood cell membrane impact of drug transporters

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Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality

International audienceBecause of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (C(blood)) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (C(PBMC)) could improve patient outcomes. The poor correlation between C(blood) and C(PBMC) makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC C(PBMC/blood) ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC C(PBMC) in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the C(PBMC/blood) in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane